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Carbon Emissions of the Tourism Telecoupling System: Theoretical Framework, Model Specification and Synthesis Effects Citation: Duan, X.; Zhang, J.; Sun, P.; Zhang, H.; Wang, C.; Sun, Y.-Y.; Lenzen, M.; Malik, A.; Cao, S.; Kan, Y. Carbon Emissions of the Tourism Telecoupling System: Theoretical Framework, Model Specification and Synthesis Effects. Int. J. Environ. Res. # Introduction Tourism development is accompanied by large-scale, fast-growing and multiple flows of people, material, capital and service, which exerts major and complex impacts on the social economy and ecological environment from the local to the global [bib_ref] Leap-Based Greenhouse Gases Emissions Peak and Low Carbon Pathways in China's Tourist..., Liu [/bib_ref] [bib_ref] Coupling Coordination and Influencing Factors among Tourism Carbon Emission, Tourism Economic and..., Pan [/bib_ref]. From 2000 to 2018, the annual growth rate of global international tourism revenues was approximately 12%, reaching US $1.5 trillion, and as many as 1.4 billion tourists were involved . International tourism growth continues to outpace the global economy. Meanwhile, the significant impacts of tourism development on ecological environment have also received great attention. In the context of people's pursuit of a comfortable, fast and long-distance tourism, tourism is no longer a smokeless industry in our imagination. In fact, tourism sectors consume a lot of energy and emit vast quantities of greenhouse gases. As a key indicator of the environmental impacts of tourism, carbon emissions have been widely considered and examined in multiple levels, such as global, national and regional [bib_ref] Global Environmental Consequences of Tourism, Gössling [/bib_ref] [bib_ref] Modeling the Dynamic Linkage between Tourism Development, Technological Innovation, Urbanization and Environmental..., Chenghu [/bib_ref]. However, previous work at different levels focused on tourism carbon emissions within a particular area, with little attention paid to tourism-integrated systems, and it especially overlooked that of tourism origins [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref] [bib_ref] Drivers of Carbon Emissions in China's Tourism Industry, Luo [/bib_ref]. The long-distance flows of people, material and energy caused by international tourism exert profound impacts on global sustainability, which are the typical issues of telecoupling. Liu et al. constructed a theoretical framework of telecoupling, which is defined as an umbrella concept that refers to the various socioeconomic and environmental interactions over distances. It extends the concept of coupled human and natural systems in time and space and can help to explore the hidden elements in it. Hence, the theoretical framework has gradually been applied in some fields, such as land use change [bib_ref] Adaptation of Global Land Use and Management Intensity to Changes in Climate..., Alexander [/bib_ref] , water resource management [bib_ref] Drought Impacts to Water Footprints and Virtual Water Transfers of the Central..., Marston [/bib_ref] , ecosystem services [bib_ref] Framing Ecosystem Services in the Telecoupled Anthropocene, Liu [/bib_ref] , international trade [bib_ref] Transatlantic Wood Pellet Trade Demonstrates Telecoupled Benefits, Parish [/bib_ref] [bib_ref] Trade in the Telecoupling Framework: Evidence from the Metals Industry, Xiong [/bib_ref] and biological diversity. Tourism is also a typical phenomenon of remote human-environment interactions. The perspective of telecoupling can help to explore the hidden elements (such as the diverse influences on tourism origins and related regions) in tourism systems. However, insufficient attention is paid to telecoupling in tourism, not to mention that in tourism environment effects. Given the above considerations, this paper intends to explore the issues of tourism telecoupling in the dimension of carbon emissions. The specific aims of this study are to (1) construct a theoretical framework of the tourism telecoupling system, (2) establish the models of tourism carbon emissions from the perspective of a telecoupling system, and (3) taking China as the example, explore the synthesis effects of international tourism carbon emissions under this framework. There are two main contributions of this study. (1) First, it builds the theoretical framework of tourism telecoupling system and constructs the calculation model of tourism telecoupling system emissions, which expand the perspective and content of tourism system research. [bib_ref] Coupling Coordination and Influencing Factors among Tourism Carbon Emission, Tourism Economic and..., Pan [/bib_ref] Two important variables in tourism carbon emission system are identified, which are the emission reduction in the origins and the implied carbon trading in tourism flows. These help to improve the understanding of tourism system carbon emissions. ## Literature ## Tourism carbon emissions at different scales Previous studies on tourism carbon emissions mainly focused on global, national and regional levels [bib_ref] Tourism Carbon Footprint Inventories: A Review of the Environmentally Extended Input-Output Approach, Sun [/bib_ref]. On the global scale, Gössling and the WTO-UNEP-WMO estimated the emissions originating from main tourism sectors (transport, accommodation and activities) based on inventory analysis. The results showed that the tourism sector accounted for approximately 3-5% of global carbon emissions in 2003 and 2005 [bib_ref] Tourism Carbon Footprint Inventories: A Review of the Environmentally Extended Input-Output Approach, Sun [/bib_ref]. Then, input-output models have emerged as a new macro-level approach to estimate tourism carbon emissions. Based on this method, the unexpected result is that the global carbon footprint of tourism was 4.5 GtCO 2 e in 2013, which is four times greater than that previously reported [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref]. The research of the global scale is important not only to improve the understanding of tourism carbon emissions but also to formulate the global emission reduction target because of the issues with long-distance international transport [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref]. On the national level, top-down and bottom-up approaches were the most commonly used [bib_ref] Measuring National Carbon Dioxide Emissions from Tourism as a Key Step towards..., Becken [/bib_ref]. The research perspectives of this level are manifold. Becken and Patterson [bib_ref] Measuring National Carbon Dioxide Emissions from Tourism as a Key Step towards..., Becken [/bib_ref] calculated CO 2 e emissions by analyzing tourism energy consumption and tourist behavior in New Zealand. Cadarso et al. estimated the contributions of residents and visitors to the tourism's carbon footprint in Spain, and Meng et al. quantified the direct and indirect CO 2 emissions of tourism of China [bib_ref] Quantifying Spanish Tourism's Carbon Footprint: The Contributions of Residents and Visitors: A..., Cadarso [/bib_ref] [bib_ref] Quantifying Direct and Indirect Carbon Dioxide Emissions of the Chinese Tourism Industry, Meng [/bib_ref]. In general, targeting the measurement of carbon emissions of tourism at the national level, existing research is mostly based on two theories: life cycle assessment and input-output, which essentially aim to measure carbon emissions from the perspectives of "consumption" and "production". National-scale studies are the key to manage the emissions of tourism industry due to the huge size of domestic travel [bib_ref] Estimating the Carbon Footprint of Australian Tourism, Dwyer [/bib_ref]. Some research have been conducted in the last decades, but the cases are mainly in developed countries [bib_ref] Tourism and Carbon Emissions: A Bibliometric Review of the Last Three Decades:..., Mishra [/bib_ref] [bib_ref] The Carbon Dioxide Neutralizing Effect of Energy Innovation on International Tourism in..., Balsalobre-Lorente [/bib_ref] [bib_ref] Carbon Intensity of Tourism in Austria: Estimates and Policy Implications, Neger [/bib_ref] [bib_ref] The Role of Tourism, Trade, Renewable Energy Use and Carbon Dioxide Emissions..., Balsalobre-Lorente [/bib_ref] and there are no details of China's international tourism using input-out method. Moreover, the research on the regional level tends to pay attention to environmental vulnerability. Sun found that the island destination leads to a larger share of carbon emissions outside their geographic territory, and Farreny et al. highlighted the significant effects of transport on tourism carbon emissions in Antarctica; approximately 70% are attributable to cruising and 30% to flying [bib_ref] Carbon Dioxide Emissions of Antarctic Tourism, Farreny [/bib_ref]. These studies indicated that tourism greatly affects the sustainability of ecologically vulnerable areas, such as nature reserves and islands. However, these studies of tourism carbon emissions at different scales focus on a particular area, with little attention paid to tourism-integrated systems [bib_ref] Impacts of Environmental Regulations on Tourism Carbon Emissions, Chen [/bib_ref] [bib_ref] The Carbon Emission Reduction Effect of Tourism Economy and Its Formation Mechanism:..., Tong [/bib_ref]. For example, the departure of tourists would reduce the carbon emissions of the origins. Tourism, especially international tourism, involves diverse and complex human-environmental interactions. The research of tourism-integrated systems should be conducted in the view of remote coupling, and the perspective of telecoupling could be an extension of global, national and regional scales. ## Telecoupling In recent decades, increased human interactions over long distances have profoundly impacted global socioeconomic and environmental sustainability. Traditional oneplace, one-way, or one-scale studies cannot analyze systematically the various social and environmental issues caused by international trade [bib_ref] Will EU Biofuel Policies Affect Global Agricultural Markets?, Banse [/bib_ref]. To address this challenge, Liu et al.proposed the framework of the telecoupling system to explore the coupling mechanism considering multiple places and directions across time and space, which reveals the socioeconomic and environmental interactions between coupled human and natural systems over distances. The research framework is composed of five major, interrelated and structured components, which are system, flow, cause, agent and effect. At the telecoupling system level, the framework includes a set of interacting coupled human and natural systems (according to the moving direction of flow, they are divided into three sub-systems: the sending system, receiving system and spillover system) through flows. At the sub-system level, every system consists of three interrelated components: agents, causes, and effects. This work expands the research scope of the interaction between remote areas and supplements the research program of complex problems with multi-scale and multi-system approaches. Therefore, the telecoupling framework provides a more comprehensive explanation of distant human-environment interactions and makes key implications for global sustainability. As a universal paradigm, the telecoupling system has been implemented in diverse fields, such as international trade [bib_ref] The Emerging Soybean Production Frontier in Southern Africa: Conservation Challenges and the..., Gasparri [/bib_ref] [bib_ref] Importing Food Damages Domestic Environment: Evidence from Global Soybean Trade, Sun [/bib_ref] , resource flow, species migration [bib_ref] Operationalizing the Telecoupling Framework for Migratory Species Using the Spatial Subsidies Approach..., Lopez-Hoffman [/bib_ref] [bib_ref] Ecosystem Service Flows from a Migratory Species: Spatial Subsidies of the Northern..., Bagstad [/bib_ref] [bib_ref] Telecoupling Framework for Research on Migratory Species in the Anthropocene, Hulina [/bib_ref] , and land use change [bib_ref] Measuring Telecouplings in the Global Land System: A Review and Comparative Evaluation..., Bruckner [/bib_ref] [bib_ref] International Progress and Evaluation on Interactive Coupling Effects between Urbanization and the..., Fang [/bib_ref]. These studies applied the telecoupling framework to explore the hidden elements within a single system and between multiple systems, and they provided some new explanations and made contributions to local and global sustainability management. In regard to tourism, Liu et al.examined the telecoupled relationships between the Wolong Nature Reserve and the rest of the world, and they treated tourism as an input flow, which has a significant effect on the natural environment. Chung et al. [bib_ref] Global Relationships between Biodiversity and Nature-Based Tourism in Protected Areas, Chung [/bib_ref] explored the relationships between biodiversity and nature-based tourism, with the conclusion that each 1% increase in biodiversity makes a 0.87% increase in tourism in a global context. Furthermore, Hulina et al. [bib_ref] Telecoupling Framework for Research on Migratory Species in the Anthropocene, Hulina [/bib_ref] applied the telecoupling framework to Kirtland's warblers (a conservation-reliant migratory songbird) and analyzed the role of tourism and other human factors in the conservation of migratory species. However, the studies of tourism telecoupling, which mainly make tourism as a factor or tourism destination as an object, remain at the exploratory stage. The theory and some other key contents of tourism telecoupling framework are still unexplored. ## Research design This study intends to discuss the tourism telecoupling system from the perspective of carbon emissions. China's international tourism is adopted as an example to estimate the carbon emissions of the tourism telecoupling systems, which include the inbound tourism, outbound tourism and the whole sectors of international tourism. There are two reasons for choosing the international tourism of China as the case. First, international tourism, as a global activity, is a typical telecoupled phenomenon. Second, the international tourism of China has a large scale, and its partners are distributed worldwide. In recent years, the scale of the inbound and outbound tourism sectors in China has been among the highest in the world, and there exists a stable two-way tourists flow between China and the 189 international tourism partners. Hence, its representativeness and typicality are both significant. ## Theoretical framework of the tourism telecoupling system The telecoupling framework provides a new perspective for the discussion of tourism system carbon emission. With the deepening of globalization and regional integration, the tourism industry in a given country is increasingly dependent on regions over distances, involving merchandise trade and personnel mobility. Therefore, the telecoupling under the tourism scenario needs to consider the resource supplier system, especially the island tourism destination, which is dependent a lot on the import of various resources. Therefore, this paper improves the telecoupling framework proposed by Liu et al.in a tourism context and constructs the tourism telecoupling system, in which a resource supplier system is called the indirect spillover system. So, the tourism telecoupling system in this study includes the sending system, receiving system, direct spillover system and indirect spillover system [fig_ref] Figure 1: Framework of the tourism telecoupling system [/fig_ref] [bib_ref] Integrated Assessments of Payments for Ecosystem Services Programs, Liu [/bib_ref]. The different subsystems interact and influence each other through tourist flows, material flows, information flows and capital flows. Specifically, taking tourism carbon emissions as an example, the sending system is the tourist origin, where the residents leave for tourism motives (causes) and then reduce its resources consumption and carbon emissions. The receiving system is a certain destination, where tourists cause additional carbon emissions. The direct spillover system is the transit system, which leads to the main environmental issues in tourism sectors [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref]. The indirect spillover system is the resource suppliers of the receiving system, which are indirectly affected by the tourism activities at destination. This tourism telecoupling system framework can not only discuss the environmental impact of tourism but also works in social, cultural and economic issues. In the international two-way tourism flow, the sending system is also a receiving system. Hence, this framework is systematic and dynamic. ## Research design This study intends to discuss the tourism telecoupling system from the perspective of carbon emissions. China's international tourism is adopted as an example to estimate the carbon emissions of the tourism telecoupling systems, which include the inbound tourism, outbound tourism and the whole sectors of international tourism. There are two reasons for choosing the international tourism of China as the case. First, international tourism, as a global activity, is a typical telecoupled phenomenon. Second, the international tourism of China has a large scale, and its partners are distributed worldwide. In recent years, the scale of the inbound and outbound tourism sectors in China has been among the highest in the world, and there exists a stable two-way tourists flow between China and the 189 international tourism partners. Hence, its representativeness and typicality are both significant. ## Theoretical framework of the tourism telecoupling system The telecoupling framework provides a new perspective for the discussion of tourism system carbon emission. With the deepening of globalization and regional integration, the tourism industry in a given country is increasingly dependent on regions over distances, involving merchandise trade and personnel mobility. Therefore, the telecoupling under the tourism scenario needs to consider the resource supplier system, especially the island tourism destination, which is dependent a lot on the import of various resources. Therefore, this paper improves the telecoupling framework proposed by Liu et al.in a tourism context and constructs the tourism telecoupling system, in which a resource supplier system is called the indirect spillover system. So, the tourism telecoupling system in this study includes the sending system, receiving system, direct spillover system and indirect spillover system [fig_ref] Figure 1: Framework of the tourism telecoupling system [/fig_ref] [bib_ref] Integrated Assessments of Payments for Ecosystem Services Programs, Liu [/bib_ref]. The different subsystems interact and influence each other through tourist flows, material flows, information flows and capital flows. Specifically, taking tourism carbon emissions as an example, the sending system is the tourist origin, where the residents leave for tourism motives (causes) and then reduce its resources consumption and carbon emissions. The receiving system is a certain destination, where tourists cause additional carbon emissions. The direct spillover system is the transit system, which leads to the main environmental issues in tourism sectors [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref]. The indirect spillover system is the resource suppliers of the receiving system, which are indirectly affected by the tourism activities at destination. This tourism telecoupling system framework can not only discuss the environmental impact of tourism but also works in social, cultural and economic issues. In the international two-way tourism flow, the sending system is also a receiving system. Hence, this framework is systematic and dynamic. ## Model specification of tourism telecoupling system carbon emissions In the context of tourism carbon emission, there is actually implied carbon trading in international two-way tourism flow. In the view of tourism telecoupling system, the reduction in the sending system carbon emission makes these synthesis effects more complex and more interesting. Therefore, the calculation of tourism telecoupling system carbon emissions contains six parts: the sending system, the receiving system, the direct spillover system and the indirect spillover system, the total carbon emissions of the tourism telecoupling system and the implied carbon trading of tourism. In this study, the carbon emissions are the CO 2 emissions stemming from energy consumption [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref] [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref]. The model specifications of every single part are described as follows. (1) The sending system carbon emissions (SS). This part includes the CO 2 emissions reduction in the sending system, resulting from residents' departing for travel. Based on the statistics of the International Energy Agency (IEA), the daily CO 2 emissions of residents include those of electricity and heat production, manufacturing industries and construction, transport, residential, commercial, and public services. The model of SS is as follows: [formula] SS = T ij ·EP i ·t ij(1) [/formula] where i and j are the origin and destination, respectively, T ij is the arrival of the destination or the departure of the origin, EP i is the per capita daily CO 2 emissions of tourists' residences, including emissions of electricity and heat production, manufacturing industries and construction, transport, residential, commercial, and public services at residences from fossil fuels, and t ij is the tourists' average length away from their origin. However, people generally leave their refrigerators on and even some other facilities causing stray currents in their homes, and this would make the results a little overestimated. From another point of view, the results would be a little underestimated, because the major international tourists are those with higher incomes, and their emissions per capita are above average. Therefore, it is considered as a reasonable estimate on the whole. (2) The receiving system carbon emissions (RS). This includes the CO 2 emissions generated by tourists at destinations. Studies on the carbon emissions of destinations are relatively mature, and the input-output approach is often used. So, this study adopts an environmentally extended input-output approach used in an interesting research made by Sun et al. [bib_ref] Tourism Carbon Footprint Inventories: A Review of the Environmentally Extended Input-Output Approach, Sun [/bib_ref]. In this method, detailed Tourism Satellite Account (TSA) is combined with global MRIO and CO 2 emissions databases, thereby tracing the CO 2 emissions originating from the consumption of tourist products and services. The model of RS is as follows [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref] [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref] : [formula] RS = q I − Tx −1 −1 y (2) [/formula] where q is a 1 × N matrix of the carbon emissions intensity, I − Tx −1 −1 is the Leontief inverse matrix, I is an N × N identity matrix, T is an N × N MRIO matrix listing the international trade transactions between countries, x = T1 T + y1 y is the total economic output, and y is an N × M matrix of the final demand of M global agents (households, governments, capital sector, and stocks) of N products. y is an N × 1 matrix acting as the final demand block of the MRIO system. (3) The direct spillover system carbon emissions (DSS). The DSS contains the emissions of transportation from the origins to the destinations, which is the key part of tourism emissions [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref]. Referring to the previous literature, this study assumes that all international tourists travel by air [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref]. The DSS model is as follows: [formula] DSS = T ij ·d ij ·k ij(3) [/formula] where T ij is arrival of tourists to a destination, d ij is the actual round flight distance from the origin to the destination, and k ij is the emission factor per unit distance [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref]. (4) The indirect spillover system carbon emissions (ISS). According to the statistics of the International Energy Agency, the energy industry (energy exploration) is the biggest contributor to carbon emissions of global trade. Hence, the ISS in this study is the CO 2 emissions originating from fossil fuels exploitation by the resource supplier. [formula] ISS = EI j ·m j ·w j (4) [/formula] where EI j denotes the energy supply quantity of the resource supplier, m j denotes the carbon emissions per unit of energy extraction by the resource supplier, and w j denotes the proportion of the energy consumption of the tourism industry in the total energy consumption of the destination [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref] [bib_ref] Inter-Market Variability in CO 2 Emission-Intensities in Tourism: Implications for Destination Marketing..., Gössling [/bib_ref] [bib_ref] Human-Environmental Relations with Tourism, Gössling [/bib_ref].The carbon emissions of the tourism telecoupling system (TSS). The TSS is the actual emissions of a certain tourism system. Hence, the TSS would be the sum of RS, DSS, and ISS minus SS. [formula] TTS = RS + DSS + ISS − SS (5) (6) [/formula] The implied carbon trading of tourism (ICT). Two-way tourism flows between countries/regions not only include economic transaction but also imply spatial transfer of the environmental responsibility. Suppose two countries A and B; the two-way tourism flows make A and B both the sending system and receiving system. The ICT is the difference value between the change in the tourism carbon emissions of country A (the RS A minus the SS A ) and the change in the tourism carbon emissions of country B (the RS B minus the SS B ). [formula] ICT = (RS A − SS A ) − (RS B − SS B )(6) [/formula] where RS A is that when A is the receiving system, SS A is that when A is the sending system, RS B is that when B is the receiving system, and SS B is that when B is the sending system. When ICT > 0, A has an implied carbon trading surplus with B, and when ICT < 0, A has an implied carbon trading deficit with B. When ICT = 0, A has an implied carbon trading balance with B. ## Data resources The data of this study come from multiple sources. The first part is tourism-related data. (1) The data of inbound and outbound tourists of China are from the World Tourism Organization. A spot of missing data is supplemented by the China Tourism Statistical Yearbook. The data of tourism satellite accounts are from the World Tourism Organization, Organization for Economic Co-operation and Development, European Union, governmental reports, and journal articles. (3) The length of stay of inbound tourists is obtained from the Tourism Sample Survey of China, while that of outbound tourists is from online travel notes. More than 3000 notes from two major tourism websites (http://www.mafengwo.cn, accessed on 12 January 2020; https://www.ctrip.com, accessed on 12 January 2020) in China were captured and analyzed to ensure information saturation. (4) The distance from the origins to the destinations considers the actual distance of round direct flight route between their capitals. The emission factor per unit distance is from Gössling and Peeters [bib_ref] Assessing Tourism's Global Environmental Impact 1900, Gössling [/bib_ref]. The second part is energy-related data. (1) The energy supply quantity of the resource supplier is from the International Energy Agency. The daily carbon emissions of residents and the carbon emissions of energy extraction by the resource supplier are from the IEA annual report (http://data.IEA.org/, accessed on 10 January 2020), which is consistent with the calculation method of IPCC. (2) The tourism energy consumption coefficient is from a journal article [bib_ref] Inter-Market Variability in CO 2 Emission-Intensities in Tourism: Implications for Destination Marketing..., Gössling [/bib_ref]. The third part is economy-related data. Global multiregional input-output tables are from the EORA database (https://worldmrio.com/eora/, accessed on 10 January 2020). It should be noted that since the maintenance and updating of tourism satellite accounts in various countries around the world are not synchronized, the carbon emissions of China's international tourism are estimated only in 2015. # Results ## Inbound tourism telecoupling system ## Sending system The CO 2 emissions of the sending system reached 5.31 Mt in 2015; namely, the inbound tourists in China reduced the emissions of their origins by 5.31 MtCO 2 , accounting for 0.28% of the global residential carbon emissions in the same year [bib_ref] International Progress and Evaluation on Interactive Coupling Effects between Urbanization and the..., Fang [/bib_ref]. Considering that this is only one country/destination, the figure is considerable. Moreover, the total and per capita CO 2 emissions of the sending system show significant spatial heterogeneity. In terms of the total emissions, the United States ranked first, reaching 1.04 MtCO 2 , accounting for approximately one-fifth of the sending system emissions, which was followed by Korea (0.87 MtCO 2 ) and Japan (0.43 MtCO 2 ) [fig_ref] Figure 2: The sending system carbon emissions of China's inbound tourism in 2015 [/fig_ref]. On a per capita basis, the emissions of developed countries are much higher than those of other countries. The per capita emissions of the United States (0.45 tCO 2 /capita), Canada (0.41 tCO 2 /capita) and Australia (0.36 tCO 2 /capita) are more than 20 times higher than those of developing countries, such as the Philippines (0.02 tCO 2 /capita), Cambodia (0.02 tCO 2 /capita) and Myanmar (0.02 tCO 2 /capita). # Results ## Inbound tourism telecoupling system ## Sending system The CO2 emissions of the sending system reached 5.31 Mt in 2015; namely, the bound tourists in China reduced the emissions of their origins by 5.31 MtCO2, accoun for 0.28% of the global residential carbon emissions in the same year [bib_ref] International Progress and Evaluation on Interactive Coupling Effects between Urbanization and the..., Fang [/bib_ref]. Conside that this is only one country/destination, the figure is considerable. Moreover, the t and per capita CO2 emissions of the sending system show significant spatial heterog ity. In terms of the total emissions, the United States ranked first, reaching 1.04 MtC accounting for approximately one-fifth of the sending system emissions, which was lowed by Korea (0.87 MtCO2) and Japan (0.43 MtCO2) [fig_ref] Figure 2: The sending system carbon emissions of China's inbound tourism in 2015 [/fig_ref]. On a per capita basis emissions of developed countries are much higher than those of other countries. The capita emissions of the United States (0.45 tCO2/capita), Canada (0.41 tCO2/capita) Australia (0.36 tCO2/capita) are more than 20 times higher than those of developing co tries, such as the Philippines (0.02 tCO2/capita), Cambodia (0.02 tCO2/capita) and My mar (0.02 tCO2/capita). ## Receiving system The total carbon emissions of the receiving system are 17.01 Mt, i.e., inbound tou produce 17.01 MtCO2 in China. On one hand, sending system emissions account for ab 20% of that of receiving system. This shows that the estimations of the tourism sys emissions in the past literature are too high. On another hand, the carbon emissio destinations is more than five times that of in origins. In other words, people's car emissions from tourism activities are more than five times those from daily activi Therefore, it is of great importance to encourage low-carbon tourism behavior. Regard the total CO2 emissions of different origins, the total emissions of Pacific Rim coun are generally higher [fig_ref] Figure 3: The receiving system carbon emissions of China's inbound tourism in 2015 [/fig_ref] ## Receiving system The total carbon emissions of the receiving system are 17.01 Mt, i.e., inbound tourists produce 17.01 MtCO 2 in China. On one hand, sending system emissions account for about 20% of that of receiving system. This shows that the estimations of the tourism system emissions in the past literature are too high. On another hand, the carbon emission in destinations is more than five times that of in origins. In other words, people's carbon emissions from tourism activities are more than five times those from daily activities. Therefore, it is of great importance to encourage low-carbon tourism behavior. Regarding the total CO 2 emissions of different origins, the total emissions of Pacific Rim countries are generally higher [fig_ref] Figure 3: The receiving system carbon emissions of China's inbound tourism in 2015 [/fig_ref] ## Direct spillover system and indirect spillover system The total CO2 emissions of the spillover system (direct spillover system and ind spillover system) are 63.08 Mt. This part is the largest of the inbound tourism telecoup system of China, and it is more than triple those of the receiving system (17.01 Mt). spatial transfer of the environmental responsibility caused by tourism development not be ignored. In detail, the carbon emissions of the direct spillover system accoun 61.38% (38.72 Mt). Moreover, the CO2 emissions of the direct spillover system (inte tional transportation) are significantly higher than those of destinations, which is sistent with the previous studies [bib_ref] Global Environmental Consequences of Tourism, Gössling [/bib_ref]. On the other hand, the indirect spillover sys emissions reach as high as 24.36 Mt, which shows the proposal of an indirect spill system is necessary and important to improve the research on tourism system emissi ## Outbound tourism telecoupling system ## Sending system In terms of outbound tourism, the sending system emissions reached 14.10 M 2015. This shows that the outbound tourists reduce Chinese emissions by 14.10 MtC which are approximately three times higher than the sending system emissions o bound tourists. This accounts for 3.93% of the Chinese total residential carbon emiss in 2015. Specifically, as shown in [fig_ref] Figure 4: The sending system carbon emissions of China's outbound tourism in 2015 [/fig_ref] , Thailand (2.10 Mt), Japan (0.80 Mt) United States (0.66 MtCO2) are the three largest countries that contributed to the send system emissions of Chinese outbound tourism. Thailand is the preferred destination Chinese residents to travel abroad in these years. On a per capita basis, Canada ( tCO2), Australia (0.22 tCO2), United States (0.21 tCO2), Italy (0.20 tCO2) and France ( tCO2) rank in the first five countries because of their longer distance from China and ple's longer stay duration. Meanwhile, those of Singapore, Korea and Japan are below of them. ## Direct spillover system and indirect spillover system The total CO 2 emissions of the spillover system (direct spillover system and indirect spillover system) are 63.08 Mt. This part is the largest of the inbound tourism telecoupling system of China, and it is more than triple those of the receiving system (17.01 Mt). The spatial transfer of the environmental responsibility caused by tourism development cannot be ignored. In detail, the carbon emissions of the direct spillover system account for 61.38% . Moreover, the CO 2 emissions of the direct spillover system (international transportation) are significantly higher than those of destinations, which is consistent with the previous studies [bib_ref] Global Environmental Consequences of Tourism, Gössling [/bib_ref]. On the other hand, the indirect spillover system emissions reach as high as 24.36 Mt, which shows the proposal of an indirect spillover system is necessary and important to improve the research on tourism system emissions. ## Outbound tourism telecoupling system ## Sending system In terms of outbound tourism, the sending system emissions reached 14.10 Mt in 2015. This shows that the outbound tourists reduce Chinese emissions by 14.10 MtCO 2 , which are approximately three times higher than the sending system emissions of inbound tourists. This accounts for 3.93% of the Chinese total residential carbon emissions in 2015. Specifically, as shown in [fig_ref] Figure 4: The sending system carbon emissions of China's outbound tourism in 2015 [/fig_ref] , Thailand (2.10 Mt), Japan (0.80 Mt) and United States (0.66 MtCO 2 ) are the three largest countries that contributed to the sending system emissions of Chinese outbound tourism. Thailand is the preferred destination for Chinese residents to travel abroad in these years. On a per capita basis, Canada (0.23 tCO 2 ), Australia (0.22 tCO 2 ), United States (0.21 tCO 2 ), Italy (0.20 tCO 2 ) and France (0.19 tCO 2 ) rank in the first five countries because of their longer distance from China and people's longer stay duration. Meanwhile, those of Singapore, Korea and Japan are below half of them. ## Receiving system The receiving system emissions reach as high as 21.60 Mt, which means Chinese tourists emitted 21.60 Mt CO2 in their destinations. This number is 1.53 times those saved in the sending system. Meanwhile, this ratio is 3.20 in Chinese inbound tourism. This shows that under the existing conditions, the energy consumption of Chinese residents' tourism activities and daily activities is closer. In terms of specific countries/regions, as shown in [fig_ref] Figure 5: The receiving system carbon emissions of China's outbound tourism in 2015 [/fig_ref] , Thailand (4.57 tCO2), Korea (2.64 tCO2), Singapore (1.38 tCO2), Vietnam (1.16 tCO2) and the United States (1.14 tCO2) are the five largest in the 189 destinations. The receiving system emissions of Thailand stand head and shoulders above others. The per capita emissions of the receiving system show similar characteristics with those of the sending system. ## Receiving system The receiving system emissions reach as high as 21.60 Mt, which means Chinese tourists emitted 21.60 Mt CO 2 in their destinations. This number is 1.53 times those saved in the sending system. Meanwhile, this ratio is 3.20 in Chinese inbound tourism. This shows that under the existing conditions, the energy consumption of Chinese residents' tourism activities and daily activities is closer. In terms of specific countries/regions, as shown in [fig_ref] Figure 5: The receiving system carbon emissions of China's outbound tourism in 2015 [/fig_ref] , Thailand (4.57 tCO 2 ), Korea (2.64 tCO 2 ), Singapore (1.38 tCO 2 ), Vietnam (1.16 tCO 2 ) and the United States (1.14 tCO 2 ) are the five largest in the 189 destinations. The receiving system emissions of Thailand stand head and shoulders above others. The per capita emissions of the receiving system show similar characteristics with those of the sending system. ## Receiving system The receiving system emissions reach as high as 21.60 Mt, which means Chinese tourists emitted 21.60 Mt CO2 in their destinations. This number is 1.53 times those saved in the sending system. Meanwhile, this ratio is 3.20 in Chinese inbound tourism. This shows that under the existing conditions, the energy consumption of Chinese residents' tourism activities and daily activities is closer. In terms of specific countries/regions, as shown in [fig_ref] Figure 5: The receiving system carbon emissions of China's outbound tourism in 2015 [/fig_ref] , Thailand (4.57 tCO2), Korea (2.64 tCO2), Singapore (1.38 tCO2), Vietnam (1.16 tCO2) and the United States (1.14 tCO2) are the five largest in the 189 destinations. The receiving system emissions of Thailand stand head and shoulders above others. The per capita emissions of the receiving system show similar characteristics with those of the sending system. ## Direct spillover system and indirect spillover system The carbon emissions of the spillover system (direct spillover system and indirect spillover system) of China's outbound tourism are 96.97 Mt, of which the direct spillover system emissions reach 70.68 Mt, accounting for 72.89% and the indirect spillover system emissions are 26.29 Mt, accounting for 27.11%. Similar with that of inbound tourism, the spillover system emissions (both direct spillover system and indirect spillover system) are much larger than those of the sending system and receiving system. Meanwhile, an interesting indicator also reveals the particularity of China as a destination. The ratio of spillover system emissions and the receiving system emissions in China's outbound tourism is 4.49, while that in China's inbound tourism is 3.71. This shows that the spatial transfer of the environmental responsibility caused by tourism development of China is lower than that of other destinations as a whole. This result is consistent with the fact that the self-sufficiency of China's tourism-related industries is relatively high. ## Synthesis effects of tourism telecoupling system carbon emissions ## Total carbon emissions of the tourism telecoupling system The tourism telecoupling system emissions in this study are the actual emissions of China's inbound and outbound tourism, which are the sum of the receiving system, the direct spillover system and the indirect spillover system minus the sending system. In 2015, the tourism telecoupling system emissions of China's international tourism reached 179.24 MtCO 2 (with the sending system at 19.41 MtCO 2 , the receiving system at 38.61 MtCO 2 , the direct spillover system at 109.40 MtCO 2 and the indirect spillover system at 50.65 MtCO 2 ). This result is consistent with the study of Sun et al., in which the carbon footprint of global tourism was discussed [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref]. Regardless of the sending system and indirect spillover system, the direct spillover system is 2.83 times that of the receiving system and about 73.91% of the sum of the two. This result is in line with the cognition of the academic community; that is, the carbon emission of international transportation accounts for about 70% in international tourism [4,6,21]. The sending system emissions accounts for 50.27% of the receiving system, showing the importance of emissions reduction in the origins in the tourism system. Furthermore, the spillover system emissions are as high as 4.15 times those of the receiving system. The spatial transfer of the environmental responsibility in tourism cannot be ignored. On a specific country basis, the carbon emissions of the tourism telecoupling system between China and another 189 countries showed a spatial heterogeneity. Geographical distance is the key variable of the telecoupling relationship. According to the geographical distance (GD) between the couple countries and the amount of their tourism telecoupling system emissions (TTSE), the 189 countries are divided into four categories: HL (high TTSE, long GD), HS (high TTSE, short GD), LL (low TTSE, long GD), and LS (low TTSE, short GD) [fig_ref] Figure 6: Geographical distance and carbon emissions of the tourism telecoupling system [/fig_ref]. High TTSE means above the average values of the TTSE, and the low TTSE is the inverse. Long GD and short GD are in the same way. The bubble size is the number of inbound plus outbound. [fig_ref] Figure 6: Geographical distance and carbon emissions of the tourism telecoupling system [/fig_ref] shows that the distribution basically conforms to Tobler's First Law of Geography, except for the United States. # Discussion and conclusions With the deepening of globalization and the increasing of remote interaction between people and the environment, academia is forced to rethink the research limitations of traditional tourism system carbon emissions. The telecoupling theory provides a feasible path for more systematic and comprehensive research on the topic. Therefore, this paper explores the issues of tourism telecoupling from the perspective of carbon emissions by taking China's international tourism as the example. The main findings are as follows: (1) the proposal of a tourism telecoupling system provides a new perspective for analyzing the carbon emissions of a tourism system. The sending system (origins) and indirect spillover system (resource suppliers) have been ignored in previous studies. (2) In the telecoupling system of China's international tourism, the emission reduction effect of the sending system is significant. The sending system emissions accounts for 50.27% of the receiving system(destinations), while this part has been ignored in previous research and practice. (3) The direct spillover system (transit) and indirect spillover system's spatial transfer effects of environment responsibility are significant. These two systems account for 89.29% of the total carbon emissions of the tourism telecoupling system. (4) There is a large implied carbon trade in international tourism. On the whole, China has a carbon-trading deficit in international tourism in 2015, reaching 23.26 MtCO 2 . Although there is no study that only estimates the carbon emissions of China's outbound and inbound tourism, some relevant studies can be compared. In our results, the tourism telecoupling system emissions of China's international tourism reached 179.24 MtCO 2 in 2015. According to Lenzen et al. [bib_ref] The Carbon Footprint of Global Tourism, Lenzen [/bib_ref] and Meng et al. [bib_ref] Quantifying Direct and Indirect Carbon Dioxide Emissions of the Chinese Tourism Industry, Meng [/bib_ref] , about 480.2 and 208.4 MtCO 2 have been generated from the domestic tourism, inbound tourism, consumption incurring domestically in relation to outbound travel in 2010. Both are larger than our results, which is mainly because it includes domestic tourism. At the same time, we can find there are some differences among some studies because of the different approaches and tourism system boundaries. For example, the emissions of tourism are 61.5 Mt in Australia and 1.438 Mt in New Zealand [bib_ref] Review of accounting for carbon dioxide emissions from tourism at different spatial..., Tao [/bib_ref]. Of course, there are great discrepancies in the industrial level, natural conditions, stage of development, technical level and industrial structure among different countries. This study has several theoretical and practical implications. First, the telecoupling system perspective is of benefit to the exploration of tourism systems. The tourism telecoupling system expands the perspective and content of tourism research. It is conducive to the in-depth exploration of the hidden elements in tourism systems by taking it as a continuously developing, globally expanding and dynamic complex system. Furthermore, this framework is applicable not only to the environmental issues but also to the economic, social and cultural interactions among destination, origin and other related regions. This is also a supplement to Mill and Morrison's tourism system model, Leiper's tourism geographic system model, and Mckercher's tourism complex system model [bib_ref] The Framework of Tourism. Towards a Definition of Tourism, Tourist, and the..., Leiper [/bib_ref] [bib_ref] A Chaos Approach to Tourism, Mckercher [/bib_ref]. Second, this study allows us to pay attention to the emission reduction effect of origins, the spatial spillover effect of tourism environment responsibility, and the implied carbon trading in tourist flows. Although these parts are significant in a tourism system, they have not received enough attention in the previous literature. The further discussion of these is necessary to expand and improve the tourism research. Third, in terms of practical implications, at the macro level, this study enables decision makers and managers to update their understanding of tourism system carbon emissions. The carbon emissions of a tourism system includes not only those of the destinations and the transportations but also those of the origins and resource suppliers. The strategies of tourism emissions reduction should be made from the perspective of global environmental governance. Finally, at the micro level, this study makes tourists realize the importance of low-carbon behavior for tourism emissions reduction. This study improves the macroscale understanding of governments and tourists regarding sustainable tourism. The sending system emissions account for 50.27% of the receiving system in China's international tourism, which means people's carbon emissions from tourism activities are about two times those of daily activities. Carbon taxes, low-carbon tourism industry, and responsible tourism behavior are all feasible attempts. However, there are also some limitations. Although this study hopes to calculate the carbon emissions of a tourism telecoupling system as accurately as possible, the overall estimate may be slightly larger due to the slight differences in the classification of tourism satellite accounts among countries. Meanwhile, the tourism telecoupling system emissions could be explored in a long-time series. Not only the environment interactions but also the economic, social and cultural issues should be discussed. Furthermore, the exploration of the subsystem level of the tourism telecoupling system, such as agents, causes, and effects, should be performed to improve the effectiveness of policies targeting socioeconomic and environmental sustainability from local to global levels. [fig] Figure 1: Framework of the tourism telecoupling system. [/fig] [fig] Figure 2: The sending system carbon emissions of China's inbound tourism in 2015 (20 main c tries are shown). [/fig] [fig] Figure 3: The receiving system carbon emissions of China's inbound tourism in 2015 (20 main c tries are shown). [/fig] [fig] Figure 4: The sending system carbon emissions of China's outbound tourism in 2015 (20 main countries are shown). [/fig] [fig] Figure 5: The receiving system carbon emissions of China's outbound tourism in 2015 (20 main countries are shown). [/fig] [fig] Figure 6: Geographical distance and carbon emissions of the tourism telecoupling system (20 main countries are shown).4.3.2. Implied Carbon Trading in Tourism FlowThe implied carbon trading (ICT) of China's international tourism is the net value of the change in the tourism carbon emissions of China (the RSChina minus the SSChina) and the change in the tourism carbon emissions of another 189 countries (the RSrelevant countries minus the SSrelevant countries). On the whole, China has a large carbon trading surplus in international tourism, reaching 23.26 CO2 Mt in 2015. This indicates that China exported 23.26 MtCO2 by two-way international tourism, accounting for 0.26% of the total CO2 emissions of fuel combustion in China[51]. Specifically, according to the definition of Section 3.1 above, China enjoys carbon trading surplus with 124 countries in international tourism, mainly including Thailand, Italy, France, Singapore, Malaysia, Spain, Cambodia, Vietnam, Indonesia, Germany, etc. Thailand is the largest one, asFigure 7shows. On the other hand, China has a carbon trading deficit with 65 countries, including Russian, Japan, the United States, Mongolia, Korea, Canada, India, Philippines United Kingdom, Australia, etc. These amounts are almost half those of the former. [/fig] [fig] Figure 7: Implied carbon trading of international tourism in China (20 main countries are shown). [/fig] [fig] Author: Contributions: Conceptualization, X.D., J.Z. and P.S.; Data curation, S.C. and Y.K.; Formal analysis, X.D.; Funding acquisition, J.Z., P.S. and C.W.; Methodology, X.D. and H.Z.; Project administration, X.D.; Resources, X.D.; Software, X.D.; Supervision, J.Z.; Validation, S.C. and Y.K.; Visualization, X.D. and P.S.; Writing-original draft preparation, X.D.; Writing-review and editing, J.Z., P.S., H.Z., Y.-Y.S., M.L. and A.M. All authors have read and agreed to the published version of the manuscript. Funding: This work is supported by the National Natural Science Foundation of China under Grant number 41771147, number 42001142, and number 42101218; and the Social Science Foundation of Jiangsu Province under Grant number: 19GLC012; and the Humanity and Social Science Foundation of Ministry of Education of China under Grant number: 21YJA630081. [/fig]

Intraocular lens power calculation in eyes with previous corneal refractive surgery Intraocular lens (IOL) power calculation after corneal refractive surgery (CRS) becomes an expanding challenge for ophthalmologists as more and more cataract surgeries after CRS are required. These patients typically also have high expectations as to visual performance. Conventional IOL power calculation schemes frequently provide inaccurate results in these cases. This review aims to summarize and recommend currently available IOL power calculation methods for eyes with the most common CRS methods: radial keratotomy (RK), photorefractive keratectomy (PRK), laser in situ keratomileusis (LASIK), and small incision lenticule extraction (SMILE). To this end, biometry measuring methods and IOL formulas will be explained and combinations of both are proposed. In synopsis, it is evident that the latest generation of vergence formulas exhibit favorable IOL power prediction accuracy in post-CRS eyes, even though the predictive precision of methods in eyes without CRS is not attained. Ray tracing computation, intraoperative aberrometry, and machine learning-based formulas hold potential to further improve refractive outcomes in post-CRS eyes. # Introduction The calculation of intraocular lens (IOL) power for patients with prior corneal refractive surgery (CRS) remains a challenging task for ophthalmologists. For the precise prediction of IOL power, generally (1) the axial length of the eye; (2) the corneal refractive power, deduced from its curvature; and (3) the effective lens position (ELP) need to be determined, as the so-called biometry of the eye. [bib_ref] Intra-ocular lens calculation status after corneal refractive surgery, Speicher [/bib_ref] This review will describe biometry measuring methods and IOL formulas and will recommend combinations of both, which lead to the precise IOL selection in post-CRS eyes. Furthermore, we will evaluate peer-reviewed literature on the new technology of intraoperative aberrometry (IA). As described previously, the precise description of corneal refractive power and ELP represent the greatest challenges in post-CRS eyes. [bib_ref] Intraocular lens power calculations in eyes with previous corneal refractive surgery: review..., Wang [/bib_ref] The main pitfall for the refractive power estimation is the altered relationship between anterior and posterior surfaces of the cornea after CRS, which is not accurately described by the standardized refractive index of a virgin cornea (1.3375). Furthermore, some methods for the measurement of the corneal curvature do not account for the anterior curvature variations within the center area, introduced by CRS. [bib_ref] Measuring total corneal power before and after laser in situ keratomileusis with..., Tang [/bib_ref] Commonly, in myopic post-CRS eyes, these inaccuracies will lead to an overestimation of corneal and to an underestimation of the IOL power to be implanted. To mitigate these errors, practical correction factors and regression formulas for a more precise determination of corneal power have been devised. [bib_ref] A new method of calculating intraocular lens power after photorefractive keratectomy, Rosa [/bib_ref] [bib_ref] New factor to improve reliability of the clinical history method for intraocular..., Rosa [/bib_ref] [bib_ref] Calculation of unknown preoperative K readings in postrefractive surgery patients, Rosa [/bib_ref] Furthermore, it is important to understand the evolution of corneal topography measuring methods, which eventually enabled the measurement of the posterior corneal curvature as an essential factor of the total corneal refractive power. ## Intraocular lens power calculation in eyes with previous corneal refractive surgery Commonly, the map, which depicts the corneal curvature, is referred to as corneal topography. A variety of methods have been devised to determine the corneal topography. In keratometry, which was first described more than 100 years ago by von Helmholtz, 7 the curvature of the anterior corneal surface is measured with four reflected light points, which are oriented on the spherocylindrical corneal surface. Spherocylindricity, however, is only a valid approximation for normal corneas, which limits the use of keratometers in post-CRS eyes. [bib_ref] Advances in the analysis of corneal topography, Se [/bib_ref] Photokeratoscopes project a Placido disk to the cornea, and the anterior corneal curvature is calculated from the reflection of the interspaced circles. 9,10 Photokeratoscopes marketed today include the Atlas 9000 (Carl Zeiss Meditec AG, Jena, Germany). Both keratometry and keratoscopy only provide information about the anterior corneal surface curvature, which can be converted to total corneal power by using a combined refractive index (1.3375) in normal corneas. In post-CRS eyes, however, additional information about the posterior corneal curvature is essential, since the radii of anterior and posterior curvature do not correspond equally after corneal surgery. [bib_ref] Advances in the analysis of corneal topography, Se [/bib_ref] [bib_ref] The precision and agreement of corneal thickness and keratometry measurements with SS-OCT..., Zhao [/bib_ref] Slit-scanning and Scheimpflug imaging represent later innovations, which facilitate the measurement of posterior corneal surface curvatures. In slit-scanning, slit light beams, which are projected through the cornea in defined angles, can yield anterior and posterior curvature as well as corneal thickness after computational analysis. [bib_ref] Orbscan computerized topography: attributes, applications, and limitations, Cairns [/bib_ref] Scheimpflug imaging as implemented in ophthalmic camera systems is based on a rotating camera, which takes images through predefined slits and thereby captures all in-focus images of the anterior segment. [bib_ref] Photography of the anterior eye segment according to Scheimpflug's principle: options and..., Wegener [/bib_ref] Scheimpflug imaging was demonstrated to have a higher repeatability in measuring the posterior corneal curvature compared with mere slit-scanning. [bib_ref] Posterior corneal shape: comparison of height data from 3 corneal topographers, De Jong [/bib_ref] Another imaging technique which has been reported to precisely describe anterior and posterior corneal curvature is optical coherence tomography (OCT), which describes the topography by inferring optical density from the backscatter of emitted infrared light. [bib_ref] Corneal topographic analysis in patients with keratoconus using 3-dimensional anterior segment optical..., Nakagawa [/bib_ref] Finally, IA methods have been developed, which use infrared light and interferometry to measure the optical power of the entire eye during surgery, delivering intraoperative aphakic autorefraction for IOL calculation, which could make preoperative biometry unnecessary. [bib_ref] Intraoperative optical refractive biometry for intraocular lens power estimation without axial length..., Ianchulev [/bib_ref] In classic IOL formulas, the calculation of the ELP, which indicates the position of the IOL relative to the cornea, is facilitated by utilizing the corneal refractive power. As the corneal refractive power and also anterior chamber depth, however, are altered by CRS, [bib_ref] Anterior chamber depth measurement before and after photorefractive keratectomy. Comparison between IOLmaster..., Bernardo [/bib_ref] classic IOL formulas lead to inaccurate predictions of the ELP. [bib_ref] Intraocular lens power calculation after corneal refractive surgery: Double-K method, Aramberri [/bib_ref] One way around this pitfall is the use of IOL formulas, which do not utilize corneal power to infer the ELP like the Haigis, Haigis-L, and Shammas. [bib_ref] Intraocular lens calculation after refractive surgery for myopia: Haigis-L formula, Haigis [/bib_ref] [bib_ref] No-history method of intraocular lens power calculation for cataract surgery after myopic..., Shammas [/bib_ref] Alternatively, double-K history methods have been developed, in which the corneal power prior to CRS contributes to the ELP prediction and the post-CRS corneal power adds to the IOL power calculation. [bib_ref] Intraocular lens power calculation after corneal refractive surgery: Double-K method, Aramberri [/bib_ref] Moreover, exact measurements of anterior chamber depth are necessary. As described above, the third essential component for the IOL power calculation is the axial length. In the past, ultrasound biometry, which cannot exactly determine the axial length with reference to the fovea, frequently lead to imprecise IOL power calculations. The advent of optical axial length measurements, applicable on eyes with sufficiently clear natural lens, improved the precision. Such methods are OCT as described above; partial coherence interferometry (PCI), which measures the travel time of infrared light from cornea to retina with interferometry; [bib_ref] Comparison of immersion ultrasound biometry and partial coherence interferometry for intraocular lens..., Haigis [/bib_ref] and optical low-coherence tomography (OLCR), which emits a continuous spectrum of wavelengths and thereby can provide more metrics than PCI. [bib_ref] Intraocular lens power formulas, biometry, and intraoperative aberrometry: a review, Kane [/bib_ref] Commercialized biometry devices employ different combinations of techniques to measure (a) corneal topography and (b) axial length as evident from the following selection of devices, which are frequently mentioned in this review: IOLMaster 500 IOL formulas can be grouped into vergence, ray tracing, and machine learning (ML) formulas. Some formulas have a mixed concept. Vergence formulas like Hoffer Q, [bib_ref] The Hoffer Q formula: a comparison of theoretic and regression formulas, Hoffer [/bib_ref] Holladay, [bib_ref] A three-part system for refining intraocular lens power calculations, Holladay [/bib_ref] Sanders-Retzlaff-Kraff theoretical (SRK/T), [bib_ref] Development of the SRK/T intraocular lens implant power calculation formula, Retzlaff [/bib_ref] Haigis,Potvin-Hill, [bib_ref] New algorithm for post-radial keratotomy intraocular lens power calculations based on rotating..., Potvin [/bib_ref] and Barrett 29 use biometry measures in the framework of Gaussian optics to calculate ELP and IOL power. Gaussian optics, however, have been shown to poorly approximate the optics of the real pseudophakic human eye. In contrast, modern ray tracing methods leverage on the computational power of present computers to exactly calculate the trajectories of all light rays with refractions at each optical surface using Snell's law. [bib_ref] Ray tracing for intraocular lens calculation, Preussner [/bib_ref] As it has been reported that the optics of pseudophakic eyes can precisely be measured with ray tracing, novel IOL calculation pipelines based on this technique hold great potential. [bib_ref] Comparison of OKULIX ray-tracing software with SRK-T and Hoffer-Q formula in intraocular..., Ghoreyshi [/bib_ref] One of these pipelines is Okulix (Panopsis GmbH, Mainz, Germany), which can be supplied with input from all common biometry devices (Zeiss, Haag-Streit, Oculus, and others). The Olsen 32 formula also uses ray tracing to exactly determine the ELP and to gather information about higher order aberrations in the optical system, which are then integrated with classic biometry measures. With regard to ML formulas, the Hill radialbasis-function (RBF) formula 33 relies completely on ML, whereas the Kane 34 formula features both ML and regression units. Since 2010, the American Society of Cataract and Refractive Surgery (ASCRS) maintains a publicly available online IOL calculator, which features a collection of vergence formulas and which will be referenced in this review (https://iolcalc.ascrs.org/). Radial keratotomy (RK), photorefractive keratectomy (PRK), laser in situ keratomileusis (LASIK), and small incision lenticule extraction (SMILE) are the CRS methods, which ophthalmologists will encounter most frequently today and from this time forth, when planning cataract surgery. Therefore, this review highlights IOL power calculation after these CRS procedures. The Medline database was searched electronically using text word synonyms and combinations of 'radial keratotomy', 'photorefractive keratectomy', 'laser in situ keratomileusis', 'small incision lenticule extraction', and 'lens power calculation'. The year of publication was limited to 2010 and onwards. The publication language was limited to English and German. There were 123 articles resulting from our search, which were screened by abstract. In all, 30 articles had relevant information for this article and were evaluated in fulltext. Adjunct literature was cited in order to provide background information. ## Iol power calculation after rk RK is a refractive surgery of the cornea for myopia, where radial incisions of the peripheral to mid-peripheral cornea are performed, which leads to a flattening of the central cornea. [bib_ref] Results of the prospective evaluation of radial keratotomy (PERK) study 10 years..., Waring [/bib_ref] Early descriptions of the procedure date back to 19th century in Europe; however, it was only widely adopted and refined in the United States and in Europe starting in the 1980s. [bib_ref] Results of the prospective evaluation of radial keratotomy (PERK) study 10 years..., Waring [/bib_ref] Linear regression analysis showed that there is a significant proportional relationship between the number of incisions and the flattening of both surfaces. [bib_ref] Scheimpflug camera measurement of anterior and posterior corneal curvature in eyes with..., Camellin [/bib_ref] But anterior and posterior surfaces do not deform in parallel. In fact, flattening is stronger in the posterior than in the anterior corneal curvature, which disqualifies the standard keratometric index for post-RK eyes. [bib_ref] Scheimpflug camera measurement of anterior and posterior corneal curvature in eyes with..., Camellin [/bib_ref] RK yields acceptable refractive results with 60% of patients within 1 diopter of the intended result. [bib_ref] Results of the prospective evaluation of radial keratotomy (PERK) study 10 years..., Waring [/bib_ref] [bib_ref] Three year results of refractive keratotomy using the Casebeer System, Werblin [/bib_ref] On the other hand, RK can lead to a progressive overcorrection and resulting hypermetropia. As a result of this shortcoming and with the rise of modern laser-based refractive procedures, RK has been used less frequently from the mid-1990s onwards. [bib_ref] Refractive surgery, Mcdonnell [/bib_ref] Yet, the patients, who have been operated with RK, will require cataract surgery in the next decades. In a recent retrospective study, the authors compare the performance of the Haigis with the Barrett True K (no history) formulas in post-RK eyes, and all biometries were measured with the IOLMaster 500 or 700 (Carl Zeiss Meditec AG, Jena, Germany). [bib_ref] IOL power calculation after radial keratotomy using the Haigis and Barrett True-K..., De Pinho Tavares [/bib_ref] The authors state that the Barrett formula results in a significantly smaller mean arithmetic refractive prediction error (ME) than the Haigis formula (-0.03 ± 0.96 D versus -0.29 ± 1.00 D, p < 0.001). Yet, there was no significant difference in the mean absolute refractive prediction error (MAE), and the Barrett formula tended to lead to hyperopic results in very flat corneas. [bib_ref] IOL power calculation after radial keratotomy using the Haigis and Barrett True-K..., De Pinho Tavares [/bib_ref] Another retrospective study comparing different subtypes of the Barrett True K formula with other vergence formulas reported the best results with the Barrett True K (history) formula yielding a median absolute error (MedAE) of 0.275 D. [bib_ref] Methods for intraocular lens power calculation in cataract surgery after radial keratotomy, Turnbull [/bib_ref] Of the formulas not requiring pre-RK refraction, the Haigis formula was found to be most accurate (ME = -0.006 D). It was less accuracy reported for the DK-Holladay-IOLM and Potvin-Hill formulas in this study. Corneal topography was determined with a Scheimpflugimaging Pentacam (Oculus Optikgeräte GmbH, Wetzlar, Germany) and other biometry measures with an IOLMaster 500 (Carl Zeiss Meditec AG, Jena, Germany). [bib_ref] Methods for intraocular lens power calculation in cataract surgery after radial keratotomy, Turnbull [/bib_ref] ## Iol power calculation after prk and lasik Since the advent of excimer laser treatment in refractive surgery in the late 1980s, several distinct procedures employing this technology have been devised. These have predominantly replaced RK as CRS methods. [bib_ref] Refractive surgery, Mcdonnell [/bib_ref] The most frequently used procedures have been PRK and LASIK, and therefore, there is an increasing demand for precise IOL power calculations in post-PRK and post-LASIK eyes. In PRK, a mechanical removement of just the corneal epithelium is followed by excimer laser ablation of corneal stroma. In contrast, corneal tissue comprising corneal epithelium and anterior stroma is initially cut and flapped open in LASIK. Subsequently, excimer laser ablation of corneal stroma is performed, and finally, the corneal flap is reinstalled in its initial position. [bib_ref] Photorefractive keratectomy versus laser in situ keratomileusis for moderate to high myopia, Hersh [/bib_ref] The mechanism of laser-ablationinduced refractive change is the same in both PRK and LASIK. Myopia correction can be facilitated by central stromal ablation leading to a flattening of the corneal curvature. Hyperopia correction can be accomplished by peripheral stromal ablation resulting in a steeper corneal curvature. [bib_ref] Refractive surgery, Mcdonnell [/bib_ref] Wang [bib_ref] Evaluation of total keratometry and its accuracy for intraocular lens power calculation..., Wang [/bib_ref] Considering that using the ASCRS calculator involves the manual insertion of biometry data into the web-interface, Ferguson et al. recently argued that using a biometerembedded formula, which automatically integrates biometry data and calculates the IOL power, would be advantageous. To this end, they match the accuracy of a biometer-embedded Barrett True K (no history) formula with a multiple formula approach in the ASCRS calculator for post-myopic and post-hyperopic eyes. For post-myopic eyes, the embedded Barrett True K (no history) formula led to the lowest MAE (0.36 D) followed by the Haigis-L formula (0.41 D) from the ASCRS calculator. Likewise, in post-hyperopic eyes, the Barrett True K (no history) formula produced the smallest MAE Volume 14 [bib_ref] Calculation of unknown preoperative K readings in postrefractive surgery patients, Rosa [/bib_ref] journals.sagepub.com/home/oed TherapeuTic advances in Ophthalmology (0.41 D), trailed by the ASCRS-average (0.46 D). The authors conclude that the biometer-embedded Barrett True K (no history) method performs equally to the ASCRS multiformula approach, while not involving manual data entry. [bib_ref] IOL power calculations after LASIK or PRK: Barrett true-K biometer only calculation..., Ferguson [/bib_ref] In another recent study, the authors used corneal topographies from the Cassini device (i-Optics, The Hague, the Netherlands) plugged into the Haigis formula for IOL power calculations after excimer laser CRS and compared the refractive outcomes with other no history formulas based on IOLMaster 700 topography. They found the following MedAEs for the Cassini Haigis, Barrett True K (no history), and Haigis-L approaches, respectively: 0.34 D, 0.34 D, and 0.49 D. [bib_ref] Color LED reflection topography: validation of equivalent keratometry reading for IOL power..., Fernández-Rosés [/bib_ref] Gasparian [bib_ref] Small-incision lenticule extraction, Moshirfar [/bib_ref] In SMILE, the fs laser initially creates a corneal stromal lenticule, without prior flap opening or removal of corneal epithelium. Subsequently, the fs laser introduces a peripheral corneal incision for the extraction of the lenticule, which then is facilitated manually with a forceps. So far, predominantly, myopic patients have been treated with SMILE. Yet, also the potential for correction of hyperopic refraction has been reported. [bib_ref] Small-incision lenticule extraction, Moshirfar [/bib_ref] Compared to LASIK, SMILE mitigates fibrotic response in the corneal stroma [bib_ref] Wound healing, inflammation, and corneal ultrastructure after SMILE and femtosecond laser-assisted LASIK:..., Luft [/bib_ref] and preserves more of the corneal tissue strength. [bib_ref] Mathematical model to compare the relative tensile strength of the cornea after..., Reinstein [/bib_ref] Also, SMILE leads to limited damage of corneal nerve fibers and reduced risk of dry eye following CRS. [bib_ref] Influence of femtosecond lenticule extraction and small incision lenticule extraction on corneal..., Ishii [/bib_ref] Similar to post-RK eyes, the Barrett True K (history and no history) with biometry provided by IOLMaster 500 or 700 (Carl Zeiss Meditec AG, Jena, Germany), because of high precision, has evolved to be the standard for IOL power prediction with vergence formulas in post-LASIK and post-PRK eyes during the last few years. This holds true both for post-myopic and post-hyperopic eyes. If Barrett True K is not available, the Haigis-L formula can still be recommended for myopic eyes. Novel advances in technology have made IA (ORA Alcon Laboratories, Inc., Fort Worth, TX) a viable alternative, achieving significantly lower MedAE and MAE than Barrett True K in a recent study. Yet, the required intraoperative technology is expensive. Again, the ASCRS calculator average has been described with similar performance to Barrett True K formula (no history) in post-LASIK and post-PRK eyes, but these results only apply if ample biometry data are collectively inserted into the calculator [e.g. RTVue (Optovue, Inc., Fremont, CA, USA) data plus IOLMaster 500 (Carl Zeiss Meditec AG, Jena, Germany) data plus Atlas (Carl Zeiss Meditec AG, Jena, Germany) data]. As such an aggregate of data will seldomly be available in regular clinic settings, the ASCRS calculator average should be considered with caution on a smaller biometry data basis. As SMILE has only been employed by clinicians since 2011, sufficient empirical data on IOL power prediction accuracy do not exist. As a broad usage of the technique, however, can be anticipated, the few theoretical studies existent so far are of interest. From these, a favorable accuracy of the Barrett True K (history and no history) and Masket formulas can be inferred. Furthermore, the Okulix (Panopsis, Mainz, Germany) ray tracing software based on Pentacam HR (Oculus Optikgeräte GmbH, Wetzlar, Germany) and an IOLMaster 500 (Carl Zeiss Meditec AG, Jena, Germany) biometry appears to be a precise method to determine IOL power. In summary, latest generation vergence formulas like the Barrett True K formula have increased the IOL power prediction accuracy in eyes with previous CRS. The predictive precision of eyes without CRS, however, has not yet been reached. Novel techniques like ray tracing computation of biometry data or IA hold the potential to further improve the refractive outcomes in post-CRS eyes. Ultimately, a further implementation of ML-based formulas, which have displayed great accuracy in virgin eyes, appears to be a logical step. # Declarations Ethics approval and consent to participate Not applicable.

Solving yeast jigsaw puzzles over a glass of wine ## Solving scientific puzzles Oddly, Pasteur and Spilsbury have much in common. In their indomitable quest to solve challenging problems, both were interested in uncovering every bit of detail of the "problem" and in piecing the "Big Picture" together. Their approach of seeking to understand the "fundamentals" in the context of potential application served us well through many centuries of scientific endeavour and remains the most powerful dynamo of technological and societal progress today. This statement also applies to the emerging science of synthetic biology. There is no doubt that synthetic biology technologies will be crucial to solve the puzzling challenges of a world with dwindling finite resources and a rapidly growing and ageing global population. Maximising the bioeconomy-that is, the economic activity derived from scientific advances and innovations in biotechnology and, in particular, the engineering of biology and biomanufacturing-will be one key strategy. However, assessing current bioeconomical trends and finding solutions for the grand challenges are like trying to solve a complex jigsaw puzzle without all the pieces in the box [fig_ref] Figure 1: The needs and trends associated with the growing global population require solutions... [/fig_ref]. The best approach is to frame human futures-improved quality of life-in a planetary context: a sustainable environment. Put differently, start by separating the puzzle's edges-well-being, security and sustainability-from the middle pieces, such as health, food, water, energy, employment and economy. Once the frame of the puzzle is pieced together, the middle pieces can be sorted by colour and a more complete picture emerges. These pieces include: prediction and prevention of diseases; affordable healthcare; adequate access to clean water and safe, nutritious foods; energy-rich molecules for renewable biofuels and novel bioenergy resources; bioremediation of polluted environments and improved land use; biodegradable pesticides and sustainable, environmentally friendly industrial chemicals; and continuous workforce training in biodesign and biomanufacturing for the new bioeconomy [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref]. Bio-based designing, bioengineering and advanced biomanufacturing to meet global needs depend on the development of our biological understanding and smart data-intensive technologies. The emerging discipline of biodesignsynthetic biology-builds on the rich legacies of several branches of biology-including genetics, molecular biology and systems biology-biomolecular platforms, chemical and physical sciences, mathematical and computational sciences, data science and bioinformatics, as well as engineering and information technology [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref]. The capability to engineer biology resulted in the development of high-throughput analytical technologies and rapid DNA sequencing, synthesis and editing technologies fast-tracked by automated platforms in genome foundries. These advances are making precision genome engineering faster, cheaper and more accurate by the day. In this context, the well-studied, foodgrade yeast, Saccharomyces cerevisiae, has become a legacy eukaryotic "chassis" for synthetic biology . In synthetic biology, the engineering term chassis refers to the organism that serves as a framework to physically accommodate new biological parts (genes), devices (gene networks) and modules (biosynthetic pathways) to (re)design biological systems (cells and organisms) [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref]. Saccharomyces cerevisiae has a long history as a model organism for fundamental academic research as well as being a workhorse for a wide range of industrial applications. Based on this track record, it is now the preferred "cell factory" of semisynthetic products, such as artemisinic acid, a precursor of the potent anti-malarial artemisinin, as well as food ingredients, including vanillin, resveratrol, saffron, stevia and nootkatone [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref]. The successful use of S. cerevisiae to produce these commercial products has moved synthetic biology from the "laboratory" to the "field", thereby changing the term "genetically modified organism" (GMO) to "semi-synthetic organism" (SSO) [bib_ref] Synthetic biology stretching the realms of possibility in wine yeast research, Jagtap [/bib_ref]. [formula] Building [/formula] ## Embo reports Synthetic biology and wine making Isak S Pretorius fresh pile of jigsaw pieces of a highly complex puzzle on the discussion tables of scientists, industry practitioners, policymakers, regulators, governments, consumers and society at large. One approach to help solve this puzzle is to create a fully manmade genome for S. cerevisiae so that we can better understand the biological intricacies of eukaryotic SSOs and be able to more accurately predict and control the practical outcomes of genome engineering-as opposed to individual gene-based genetic engineering. This is the primary purpose of the international Synthetic Yeast Genome Project, known as Yeast 2.0 or Sc2.0. This ambitious collaborative project is guided by an agreed and legally binding policy statement on key issues, such as social benefits, intellectual property, safety and governance [bib_ref] Design of a synthetic yeast genome, Richardson [/bib_ref] [bib_ref] Freedom and responsibility in synthetic genomics: the Synthetic Yeast Project, Silva [/bib_ref]. The laboratory benches of a dozen Sc2.0 research groups around the world (USA, UK, China, Singapore and Australia) are strewn with pieces of a complex 6,000-piece (6,000gene) jigsaw puzzle comprising the genetic make-up of S. cerevisiae [bib_ref] Design of a synthetic yeast genome, Richardson [/bib_ref]. The pieces have been sorted into 16 piles (chromosomes) by colour, shape and size and divided between the puzzle masters . The challenge is to recreate the guide picture on the front of the box-a round-to-ovoid single-celled fungus, 50-10 lm in diameter and compartmentalised like most other eukaryotic cells, including an encapsulated nucleus. While remaining true to this "blueprint", each of the collaborating laboratories must meet the challenge of designing, building and interlocking the pieces of the S. cerevisiae genome puzzle in order to, for the first time, completely rebuilt a eukaryote's genome. Each piece of the puzzle is essential if a complete picture is to be produced, and the Sc2.0 team is working to have it in place by 2018. Isak S Pretorius Synthetic biology and wine making EMBO reports be synthesised by the end of this year. The Sc2.0 project is thus on track to consolidate the 16 chemically synthesised chromosomes into a single cell of S. cerevisiae by 2018 [fig_ref] Figure 6: The collaborative Yeast 2 [/fig_ref]. This consolidated Sc2.0 genome was designed from the start to be fully customisable so that researchers will be able to ask otherwise intractable questions about the fundamentals of chromosome structure, organisation, function and evolution, as well as gene content, function of RNA splicing and the role of small RNAs in yeast biology [bib_ref] Design of a synthetic yeast genome, Richardson [/bib_ref]. The guiding design principles for the Sc2.0 genome aspired to balance a desire to preserve the phenotype of the wild-type yeast strain while incorporating inducible flexibility for further manipulation and minimising instability resulting from the repetitive nature of yeast's native genomic DNA. These principles for the design, construction, analysis, fitness testing and curation are most likely to be scalable to future synthetic work on the larger genomes of plants, animals and humans [bib_ref] Design of a synthetic yeast genome, Richardson [/bib_ref]. The final Sc2.0 genome is therefore designed, curated, streamlined and reorganised to encode a slightly modified genetic code [bib_ref] Design of a synthetic yeast genome, Richardson [/bib_ref]. To facilitate the assembly of the synthetic chromosomes, specific base substitutions within some of the open reading frames (ORFs) are included to introduce necessary or remove inconvenient enzyme recognition sites. In addition, recognisable PCRTags-short recoded sequences within certain ORFs facilitating a polymerase chain reaction (PCR)-based assay-are also included in the design of the Sc2.0 genome so that the synthetic DNA can be distinguished from native DNA [bib_ref] Design of a synthetic yeast genome, Richardson [/bib_ref]. Other important modifications include the addition of many loxPsym sites for future genome scrambling; all TAG stop codons are recoded to TAA; all repetitive and dispensable sequences, such as Ty transposons, introns, subtelomeric regions and silent mating-type loci (HML and HMR located on Chromosome 3) are omitted from the design; and all tRNA genes are relocated to a novel neochromosome [bib_ref] Design of a synthetic yeast genome, Richardson [/bib_ref]. The expectation is that these As the Sc2.0 project is progressing, genome engineering technologies are being advanced at a rapid pace while important fundamental biological intricacies of yeast cells are being figured out. By the end of this project, it would be known, for example, whether removing all introns and transposable elements will affect cell fitness and whether the relocation of all tRNA genes to a 17 th mini-neochromosome will disadvantage the genetic processes and protein synthesis machinery of the redesigned haploid S288c laboratory strain of S. cerevisiae. These are just a few examples of the puzzling questions that are being answered as the Sc2.0 picture emerges. ## Customising wine yeast under challenging conditions From a wine scientist's viewpoint, another perplexing question is whether some of fundamental insights from the Sc2.0 yeast strain can be extrapolated to robust industrial wine yeast strains [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref] [bib_ref] Yeast's balancing act between ethanol and glycerol production in low-alcohol wines, Goold [/bib_ref]. Wine yeast researchers expect to gain much knowledge by borrowing some of the Sc2.0 puzzle pieces to build their favourite wine yeast strains. Since yeast fermentation is a centre piece in the process of winemaking, there is much to be gained by unlocking the genetic secrets that make different wine yeast strains perform differently. By understanding the fundamentals, the possibilities could be expanded by redesigning some wine yeast strains' natural jigsaw puzzle pieces or inventing totally new ones. The objective of such strain development programmes would be to provide winemakers with a diverse array of wine yeast strains. Each strain would be specifically tailored to produce particular wine styles identified for various markets and market segments the world over. Not all yeast strains are equally able to catalyse rapid, complete and efficient conversion of grape sugars to ethanol, carbon dioxide and other minor, but important metabolites-acids, alcohols, carbonyls, esters, terpenes, thiols and so on without the development of off-flavours (e.g. hydrogen sulphide, volatile acids and volatile phenols) [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref] [bib_ref] Yeast's balancing act between ethanol and glycerol production in low-alcohol wines, Goold [/bib_ref]. Wine yeasts can differ widely in terms of their robustness, fermentation efficiencies and sensory properties, and performance depends on the specific composition of a particular grape juice and specific fermentation conditions and techniques used by the winemaker. During the past three decades or so, a wide variety of strain improvement techniques have been harnessed to optimise fermentation performance, robustness, spoilage control, processing efficiency, product wholesomeness and sensory quality [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref] [bib_ref] Yeast's balancing act between ethanol and glycerol production in low-alcohol wines, Goold [/bib_ref]. Non-genetic modification techniques include hybridisation (mating or crossbreeding), mutagenesis (induction of ## Embo reports Synthetic biology and wine making Isak S Pretorius mutations by exposure to mutagenic chemicals or ultraviolet radiation) and adaptive evolution (crossing and back-crossing of selected mutants) [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref]. Several hybrid and mutant strains have been used successfully in global commercial winemaking. Consumers had no hesitation embracing the many award-winning wines produced with rapidfermenting and aroma-enhancing hybrid strains originating from mating and crossbreeding, or the many fault-free wines produced with mutants that no longer produce off-flavours, such as hydrogen sulphide, volatile acidity and volatile phenols [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref] [bib_ref] Yeast's balancing act between ethanol and glycerol production in low-alcohol wines, Goold [/bib_ref]. Such broad-based acceptance by producers and consumers is, however, not the case for strains generated by genetic engineering. More than 10 years ago, the first two GM wine yeast strains, ML01 and ECMo01, which met all regulatory requirements, were commercialised in the USA, Canada and Moldova [bib_ref] Genetic engineering of an industrial strain of Saccharomyces cerevisiae for L-malic acid..., Volschenk [/bib_ref] [bib_ref] Metabolic engineering of Saccharomyces cerevisiae to minimize the production of ethyl carbamate..., Coulon [/bib_ref]. Despite the proven success in winemaking trials and the clear benefits to both producers and consumers of the ML01 malolactic strain and the ECMo01 low-ethylcarbamate strain, there is yet to be widespread uptake of these wine yeasts in commercial winemaking. ML01 and ECMo01 are not the only GM wine yeasts twiddling their budding thumbs at the entrances of wineries and cellar doors . Several robust and flavour-active strains have been developed to mitigate stuck fermentations during problematic hot vintages and to create market-driven wines with desired alcohol levels [bib_ref] Yeast's balancing act between ethanol and glycerol production in low-alcohol wines, Goold [/bib_ref] [bib_ref] Evaluation of gene modification strategies to develop low-alcohol wine yeasts, Varela [/bib_ref] and aroma profiles [bib_ref] Synthetic genome engineering forging new frontiers for wine yeast, Pretorius [/bib_ref] [bib_ref] Yeast's balancing act between ethanol and glycerol production in low-alcohol wines, Goold [/bib_ref]. So far, the well-orchestrated anti-GMO campaigns, the furore over the labelling of GM food products and associated market sensitivities have deterred winemakers to take full advantage of science and the opportunities afforded by genetic engineering, and now more recently, by genome engineering. While the wine industry is caught up in the scientific and cultural maelstrom of the "wonders and terrors" of GMOs and SSOs, researchers continue to mine DNA sequencing data for the responsible design, synthesis and/or editing of wine yeast genomes with huge potential benefits for producers and Isak S Pretorius Synthetic biology and wine making EMBO reports consumers alike. On one hand, anti-GMO/ SSO campaigners and uninformed traditionalists might dismiss such research as an "unwanted reality" that will eventually vanish into thin air. However, futurefocussed innovators, on the other hand, are highly supportive of these research efforts because they recognise that it generates invaluable insights into the molecular intricacies of wine yeast cells. Akin to what research into the Higgs boson elementary particle is revealing about the "Standard Model" of particle physics, synthetic genomic experimentation is illuminating the biomolecular mysteries of wine yeast cells. Factually correct information and knowledge gained from such fundamental research and evidence-based data are the only way to counteract ideologically driven doomsday prophecies, exaggerated fantasies, empty promises and guesswork about the future of SSOs. Uncorking a raspberry-tasting Chardonnay produced by a semi-synthetic wine yeast History was made with the recent successful development of the world's first semisynthetic wine yeast as a "demonstrator" project. A cassette of four synthetic genes encoding the production of a highly desirable fragrant raspberry ketone-4-[4-hydroxyphenyl]-butan-2-one-was embedded into the genetic blueprint of a wine yeast strain [fig_ref] Figure 8: The first semi-synthetic wine yeast capable of producing Chardonnay wine with a... [/fig_ref] [10]. This phenylpropanoid is the primary aroma compound found in several fruits, vegetables and berries, including raspberries, blackberries, grapes and rhubarb, but, owing to the low concentrations present in these plants, it is not economical to extract this flavoursome compound from its natural sources. However, thanks to market preferences, chemically manufactured derivatives of this flavouring agent fetch much lower prices than the naturally derived form. This led to early attempts to produce raspberry ketone from p-coumaric acid in heterologous bacterial and yeast strains. Yet, the high cost of p-coumaric acid as a substrate and the trace amounts of raspberry ketone obtained in these GM strains prevented commercial production of this phenylpropanoid as a food-grade flavouring agent. The missing ## Met5 met10 Deletion of the α and β [bib_ref] Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via..., Lee [/bib_ref]. Solving such a scientific puzzle starts with the unravelling of the phenylpropanoid biosynthetic pathway. This pathway commences with the conversion of phenylalanine to p-coumaric acid via cinnamate or directly from tyrosine to p-coumaric acid. Conversion of p-coumaric acid to raspberry ketone requires three additional enzymatic steps including a condensation reaction between coumaroyl-CoA and malonyl-CoA. To design a biosynthetic pathway for the de novo production of 4-[4-hydroxyphenyl]butan-2-one in a wine yeast, the following codon-optimised genes were chemically synthesised and integrated into the HO locus of a wine yeast strain (AWRI1631): the phenylalanine ammonia lyase from an oleaginous yeast, Rhodosporidium toruloides; the cinnamate-4-hydroxylase from Arabidopsis thaliana; and the coumarate CoA ligase 2 gene from parsley, Petroselinum crispum, fused by a rigid linker to the benzalacetone synthase from rhubarb, Rheum palmatum. This semisynthetic organism was equipped to produce raspberry ketone at concentrations almost two orders of magnitude above its predicted sensory threshold in Chardonnay grape juice under standard wine fermentation conditions, while retaining the ability to ferment the grape must to dryness [bib_ref] Heterologous production of raspberry ketone in the wine yeast Saccharomyces cerevisiae via..., Lee [/bib_ref]. The primary goal of this research project was not to produce raspberry-tasting Chardonnay at a commercial scale. The objective was to hone our synthetic biology skills and to expand our toolkit with which we can advance fundamental understanding and provide solutions to the many riddling questions of flavour-active wine yeast puzzles. By solving fundamental yeast jigsaw puzzles over a glass of wine, we might well acquire the ability to design the ultimate wine yeast genome model, thereby paving the way for further improvement of wine quality and consumer acceptance while minimising resource inputs, production costs and environmental impact. [fig] Figure 1: The needs and trends associated with the growing global population require solutions to a wide variety of grand challenges linked to human well-being, security and the sustainability of the environment.EMBO reports Vol 18 | No 11 | 2017 ª 2017 The Author [/fig] [fig] Figure 2: "As the Sc2.0 project is progressing, genome engineering technologies are being advanced at a rapid pace while important fundamental biological intricacies of yeast cells are being figured out." ...................................................... The design of the Sc2.0 genome draws on data from the genome sequence first announced in 1996 for a haploid laboratory strain (S288c) of S. cerevisiae. Thẽ 12 Mb (non-redundant) to~14 Mb (total) genome sequence carries approximately 6,000 genes of which about 5,000 are individually non-essential. The 6,000 genes are distributed along 16 linear chromosomes of varying length (200-2,000 kb). The first step towards designing and building the S. cerevisiae's genome was taken in 2011 with the synthesis of the two arms of Chromosome 3-the third smallest S. cerevisiae chromosome [2,3]. This opened the way for the synthesis of Chromosome 3 in full in 2014. Earlier this year, the synthetic versions of five more S. cerevisiae chromosomes have been published (Fig 5) Biodesign and biomanufacturing underpinned by synthetic biology technologies are key success factors in the new bioeconomy. ª 2017 The Author EMBO reports Vol 18 | No 11 | 2017 [/fig] [fig] S a c c h a r o m yc es c e r e v i s i a e, Figure 3: The multi-purpose yeast, Saccharomyces cerevisiae, is the best-studied eukaryotic model organism and the most used microbe in the fermentation industry.EMBO reportsVol 18 | No 11 | 2017 ª 2017 The Author EMBO reports Synthetic biology and wine making Isak S Pretorius designer changes would not cause any fitness defects but would allow a whole gamut of future genome manipulations and research opportunities. To date, about 75% of the DNA synthesis is complete and built into discrete strains by the various Sc2.0 teams. ...................................................... "History was made with the recent successful development of the world's first semisynthetic wine yeast as a "demonstrator" project. " ...................................................... [/fig] [fig] Figure 5: Key milestones in terms of the synthesis of viral and bacterial genomes inspired the idea to chemically synthesise the 16 chromosomes of the yeast Saccharomyces cerevisiae and replace the native chromosomes with the synthetic chromosomes. EMBO reports Vol 18 | No 11 | 2017 ª 2017 The Author [/fig] [fig] Figure 6: The collaborative Yeast 2.0 (Sc2.0) project, which commenced in 2011, has made significant progress with 6 of the 16 chromosomes synthesisedwell on track to have all the native chromosomes of the yeast Saccharomyces cerevisiae replaced by 2018. ª 2017 The Author EMBO reports Vol 18 | No 11 | 2017 [/fig] [fig] Figure 8: The first semi-synthetic wine yeast capable of producing Chardonnay wine with a raspberry aroma.ª 2017 The Author EMBO reports Vol 18 | No 11 | 2017 [/fig]

Spontaneous subdiaphragmatic bar migration after pectus excavatum treatment ## Summary A case of an adolescent boy with persistent thoracic symptoms and recurrence of pectus excavatum (after previous treatment with the Nuss procedure) is presented. During thoracoscopic revision, subdiaphragmatic migration of the implant was noted. The bar was removed without damage to the intraabdominal organs or other complications, and a new bar was placed and stabilised. Revision showed successful correction of the thorax, and the boy had no thoracic symptoms. # Background The Nuss procedure is the most applied surgical treatment of pectus excavatum (PE). It was developed by Donald Nuss in 1987 and implemented as primary treatment in the late 1990s. [bib_ref] Complications of the "Nuss Procedure, Garzi [/bib_ref] This minimally invasive surgery achieves improvement of thoracic anatomy, aesthetics and cardiopulmonary function. However, early and late complications of the Nuss procedure often occur. Complications may be seen intraoperatively (eg, haemothorax, pericardial perforation, arrhythmias) or postoperatively (eg, bar displacement, pleural effusion, pneumothorax, wound infections, pericardial effusion). [bib_ref] Complications of the "Nuss Procedure, Garzi [/bib_ref] Bar displacement is one of the most prevalent complications. [bib_ref] Complications of the "Nuss Procedure, Garzi [/bib_ref] Three types of bar displacement can be distinguished: lateral sliding, flipping or posterior disruption. Displacement is mostly seen within 30 days after initial placement, [bib_ref] Complications associated with the Nuss procedure: analysis of risk factors and suggested..., Park [/bib_ref] although it can occur at any point during follow-up. Fortunately, due to the introduction of stabilisers and fixation techniques, the incidence has decreased over the years from 15% to 3%-7%. [bib_ref] The search for stability: bar displacement in three series of pectus excavatum..., Tedde [/bib_ref] In this report, we present a patient with an abdominal location of a bar due to the extraordinary occurrence of subdiaphragmatic migration following the Nuss procedure. ## Case presentation The patient is an adolescent boy with a history of mild scoliosis and epilepsy, without any seizures or medication in the 7 years prior to surgery. To remedy cosmetic problems of PE, he underwent a thoracoscopic-guided Nuss procedure. During the procedure, no unexpected events occurred and the patient completed his admission without complications. Perioperative images showed a good intrathoracic placement of the Nuss bar. One month after surgery, he presented with thoracic pain, which was interpreted as persistent postoperative pain. Physical examination and a thoracic X-ray showed no abnormalities and an unaltered position of the Nuss bar. Eight months after surgery, he presented with position-dependent discomfort and a recurrence of the PE, for which bar revision was indicated. During surgery, subdiaphragmatic and intraabdominal migration of the bar were seen. Subsequently, the bar was removed, and a new bar was placed intrathoracically. ## Initial nuss bar placement The initial bar placement by video-assisted thoracoscopy (VATS) was done under both general and epidural anaesthesia, with the patient placed in supine position. The bar was measured and bent to fit size. An incision was made on both sides of the thorax, followed by submuscular tunnelling. A 5 mm trocar was introduced in the anterior section of the axillary region on the right side, followed by insufflation of carbon dioxide and tunnelling under thoracoscopic vision. A precordial tunnel from side to side was created. A silicon tube was secured to the tunneler, after which the previously bent bar was pulled back through the precordial tunnel (figure 1). The bar turned with good result, and haemodynamic changes were not noted. A fixation plate was introduced on the left side, on which the lateral part of the bar was bent to prevent slip-off of the plate. Haemostasis of the precordial tunnel and operative wounds was achieved. The wounds were closed in layers. The procedure was carried out without any complications and with minimal blood loss. The patient was discharged on postoperative day 3. ## Investigations The first postoperative X-ray showed a good position of the bar (figure 2). When thoracic symptoms persisted, a second X-ray was performed, which was initially described as showing no abnormalities (figure 3). A second reading, however, did show caudal displacement and potential rotation of the bar with the right hemidiaphragm projecting over the bar. ## Treatment The revision by VATS was carried out under both general and epidural anaesthesia, with the patient placed in supine position. Incisions were made at the same level as during the previous surgery and scar tissue was excised. Subsequently, the bar and fixation plate on the left side were mobilised. Led by palpable movement, localisation of the bar was attempted on the right side without any success. A 5 mm trocar was introduced in the frontal axillary region on the right side, followed by creating thoracoscopic vision. Under direct vision, the bar could not be identified on the right side of the thorax. Migration to the subdiaphragmatic region appeared to have occurred, as movement of the bar was seen below the diaphragm. There were no signs of damage to the diaphragm or surrounding tissues [fig_ref] Figure 4: Intraoperative view of absent intrathoracic bar with intact diaphragm [/fig_ref]. The left thoracic incision site was extended, and the Nuss bar was manually removed through the left side, without any haemodynamic changes. A precordial tunnel from side to side was created again. A silicon tube was secured to the tunnelling instrument, after which the newly bent bar was retracted through the precordial tunnel. The bar turned with good result and without any haemodynamic changes. A fixation plate was introduced; this time on the right side, on which the lateral part of the bar was bent to prevent slip-off of the plate. Haemostasis of the precordial tunnel and operative wounds was achieved. The wounds were closed in layers. The operation was carried out without Case report any complications and with minimal blood loss. The patient was discharged on postoperative day 3. ## Outcome and follow-up The postoperative course was without complications. Five days after the revision procedure, adequate correction of anatomy and appearance of the thorax were seen. The patient had no thoracic symptoms and was pleased with the result. The X-ray showed an adequate position of the bar (figure 5). # Discussion Extrathoracic or intra-abdominal migration of a Nuss bar is a rare event, in contrast to intrathoracic bar displacement, which occurs rather frequently. Several cases have been reported with different types of bar displacement and only a few with intra-abdominal migration. Simon et al have reported an intra-abdominal migration of a bar into the stomach in a man in his 20s. [bib_ref] Extra-thoracal migration of the Nuss bar in corrective surgery for pectus excavatum:..., Simon [/bib_ref] Tahmassebi et al reported a case of a man in late adolescence, in which VATS failed to view an intrathoracic bar. [bib_ref] Intra-abdominal pectus bar migration--a rare clinical entity: case report, Tahmassebi [/bib_ref] By instrumental palpation, subdiaphragmatic position of the bar was concluded. Via an incision in the diaphragm, the bar was successfully removed without complications. Moreover, Tahmassebi et al present 12 other bar migration cases from literature, in most of which the aetiology remained unclear. Migration sites included peritoneum, ascending aorta, left ventricle of the heart and liver. [bib_ref] Intra-abdominal pectus bar migration--a rare clinical entity: case report, Tahmassebi [/bib_ref] Among other types of displacement, Hoel et al reported a life-threatening laceration of the ascending aorta in a young boy 2 months after a Nuss procedure. A cardiac tamponade was the consequence of this laceration, which was relieved by immediate needle aspiration from the pericardium and subsequent median sternotomy for removal of the bar. [bib_ref] A life-threatening complication of the Nuss procedure for pectus excavatum, Hoel [/bib_ref] Furthermore, Yang et al presented a similar case in which displacement of the bar caused cardiac tamponade 6 months after the Nuss procedure. [bib_ref] Delayed cardiac tamponade after the Nuss procedure for pectus excavatum: a case..., Yang [/bib_ref] Kanegaonkar and Dussek presented a case report of three bar migration cases. One case was a patient in early adolescence who at the age of 2 years had received a lower lobectomy for an unresolving lobar consolidation. Because of sternal depression, the patient underwent resection of the costal cartilages from the third rib downwards and a partial transverse sternotomy followed by a bar placement. During elective bar removal after 2 years, a part of the bar was seen buried into newly formed bone of the sixth rib. The other two cases presented by Dussek concerned bar migration into the thoracic cavity. One was observed during elective bar removal, the other was seen on CT after a failed attempt to remove the bar because it could not be located during surgery. [bib_ref] Removal of migrating pectus bars by video-assisted thoracoscopy, Kanegaonkar [/bib_ref] While bar displacement may occur in 3%-7% of cases, bar migration is a rather extraordinary occurrence with an unknown prevalence. It could be defined as passage of foreign material through organs or tissues without any damage to those organs or tissues. [bib_ref] Intra-abdominal pectus bar migration--a rare clinical entity: case report, Tahmassebi [/bib_ref] In our case, indeed, damage to the diaphragm was seen neither during the initial procedure nor during the second procedure. Several mechanisms have been proposed that could cause such migration events, including cardiac and lung activity, patient rotation activities, narrow thickness of the bar and unilateral fixation of the bar where the movement of the contralateral side is not limited. Furthermore, in the case of bar migration to the subdiaphragmatic region, self-healing of the diaphragm could conceal the extrathoracic position of the bar during surgery. If migration of a Nuss bar is suspected, thorough examination through X-rays and, if necessary, CT scans are advocated to be the first course of action. Furthermore, an ultrasound could confirm whether the bar is located above or below the diaphragm. In addition, MRI could assess if the bar has penetrated diaphragmatic structures. When bar migration or displacement of the bar is highly suspected, but not seen on imaging techniques, a second look through thoracoscopic surgery is indicated. When intra-abdominal migration is seen on additional imaging, a revision through endoscopic surgery is necessary to safely remove the bar. Furthermore, VATS or laparoscopy may be insufficient to locate and remove the migrated bar, such as in the case described by Tahmassebi et al. In such circumstances, an open approach through laparotomy may be the best option for correct and safe removal of the Nuss bar. [bib_ref] Intra-abdominal pectus bar migration--a rare clinical entity: case report, Tahmassebi [/bib_ref] Nevertheless, novel methods have been proposed for reducing the risk of bar migration or displacement. For instance, Tam et al recently presented the ZipFix stabilisation method in 20 patients without any recurrence or bar displacement, by using a biocompatible cable-tie implant. [bib_ref] Novel Nuss bar fixation using ZipFix for pectus excavatum, Tam [/bib_ref] However, these methods are yet to be further studied for their success rates. In conclusion, bar migration following the Nuss procedure is a rare occurrence with an unknown aetiology. On occurrence or suspicion of bar migration, cautious action through [fig] Figure 1: Intraoperative view of first bar placement. [/fig] [fig] Figure 2: Postoperative X-thorax after first bar placement. [/fig] [fig] Figure 3: Follow-up X-thorax. [/fig] [fig] Figure 4: Intraoperative view of absent intrathoracic bar with intact diaphragm. [/fig] [fig] Figure 5: Postoperative X-thorax after bar revision. [/fig]

Long-term efficacy of alemtuzumab in polymyositis References1 Rovin BH, Furie R, Latinis K et al. Efficacy and safety of rituximab in patients with active proliferative lupus nephritis: the Lupus Nephritis Assessment with Rituximab study. Arthritis Rheum 2012;64:121526. 2 Merrill JT, Neuwelt CM, Wallace DJ et al. Efficacy and safety of rituximab in moderately-to-severely active systemic lupus erythematosus: the randomized, doubleblind, phase II/III systemic lupus erythematosus evaluation of rituximab trial. Arthritis Rheum 2010;62:22233. 3 Condon MB, Ashby D, Pepper RJ et al. Prospective observational single-centre cohort study to evaluate the effectiveness of treating lupus nephritis with rituximab and mycophenolate mofetil but no oral steroids. Ann Rheum Dis 2013;72:12806. 4 Mysler EF, Spindler AJ, Guzman R et al. Efficacy and safety of ocrelizumab in active proliferative lupus nephritis: results from a randomized, double-blind, phase III study. Long-term efficacy of alemtuzumab in polymyositis SIR, We present a PM patient refractory to standard therapy, who showed effective clinical remission after a single treatment cycle with alemtuzumab for >3 years of follow-up. This is, to our knowledge, the first report of long-term efficacy of alemtuzumab in the treatment of PM. This Caucasian male patient was born in 1957. PM manifested at the age of 52 with progressive symmetrical muscle weakness and myalgia of proximal limb musculature starting in 2009. At first consultation the patient showed a positive Gower's sign and maximum walking distance was reduced to 500 m. The diagnosis of PM was established in May 2009 and was based on typical clinical presentation, elevated creatine kinase (CK) levels (3237 U/ l), myopathic changes in the electromyogram and muscle biopsy showing endomysial CD8 + T cell infiltration [fig_ref] FIG. 1: Histology, clinical disease course and creatine kinase levels in a patient with... [/fig_ref]. Ro-52 autoantibodies were positive; other myositis-specific autoantibodies (anti-Jo-1, ant-Mi-2 or anti-SRP) could not be detected. Therapy with oral prednisolone (80 mg/day) in combination with azathioprine (start dose 50 mg/day, end dose 225 mg/day) was initiated, significantly ameliorating muscle weakness and myalgia. Azathioprine therapy had to be withdrawn in August 2009 due to extremely elevated liver enzymes. Under subsequent therapy with ciclosporin (200 mg/day), it was not possible to further taper the prednisolone dose (<40 mg/day). The disease course showed sustained progression, as CK levels were still considerably elevated. Thus we started i.v. cyclophosphamide (monthly cycles at 2 Â 1000 mg, followed by 2 Â 1300 mg), which was not able to slow disease progress, decrease CK level or spare corticosteroid therapy. Walking distance further deteriorated to 150 m. A combination therapy of IVIG (start dose 5 Â 40 g/day, followed by monthly cycles of 3 Â 30 g/day) and MTX (start dose 7.5 mg/day, end dose 30 mg/day) was initiated in September 2010. After an initial favourable response, the disease progressed further despite increasing MTX doses to 30 mg/day, with CK levels increasing to 5242 U/l, and the patient reported significant deterioration of muscle strength. IVIG and MTX were subsequently withdrawn. After giving informed consent, the patient received alemtuzumab (one cycle at 5 Â 30 mg) under premedication with clemastine, ranitidine, paracetamol, ondansetron and i.v. methylprednisolone (250 mg/day) in May 2011 [fig_ref] FIG. 1: Histology, clinical disease course and creatine kinase levels in a patient with... [/fig_ref]. Alemtuzumab led to a rapid and long-lasting depletion of T cells, B cells and NK cells (supplementary [fig_ref] FIG. 1: Histology, clinical disease course and creatine kinase levels in a patient with... [/fig_ref] , available at Rheumatology Online). At first application, the patient suffered from infusion-related reactions with fever and chills, while subsequent infusions were well tolerated. Afterwards the patient received famciclovir, fluconazole and cotrimoxazole for infection prophylaxis until CD4 + T cells reached >200 cells/ml. Approximately 12 weeks later the patient noticed an improvement in muscle strength, which was confirmed on physical examination and was slightly preceded by a continuous decrease in CK level. In addition, constant improvement of walking distance and prednisolone tapering to 7.5 mg/day was achieved. Until the beginning of July 2014 the disease course remained stable, when the patient reported progressive myalgia and deterioration of walking distance. No severe adverse events have been observed so far. We decided to administer another cycle of alemtuzumab. Current therapeutic options of PM consist of corticosteroids, IVIG and immunosuppressants such as AZA or MTX [bib_ref] Immunotherapy of myositis: issues, concerns and future prospects, Dalakas [/bib_ref]. These therapies are mainly non-specific, have various adverse effects and often show limited efficacy. Monoclonal antibodies are emerging as new therapeutic strategies for autoimmune myopathies, however, to date only limited evidence exists for their use [2]. Alemtuzumab is a monoclonal anti-CD52 antibody leading to rapid, long-lasting depletion of immune cells, but not of haematopoietic stem cells. After depletion, the reconstitution of immune cells follows a certain pattern, with T cells being the last to recover after years . Here, anti-CD52 treatment was initiated under the rationale that CD8 + T cells are critically involved in the progressive destruction of muscle cells in PM and are found clonally expanded in the muscle of PM patients [4]. In our standard therapyrefractory patient, we observed a stable disease course with constant improvement of muscle strength lasting for $3 years, adding to previous reports providing short-term observations of alemtuzumab efficacy in PM . It should be kept in mind, however, that interpretation of our data is limited by previous immunosuppressive medication. The high incidence of secondary autoimmunity ($30%) in alemtuzumab-treated patients should always be considered in therapeutic decisions . Thorough surveillance is needed to prevent serious adverse events after alemtuzumab infusion. In conclusion, we present the first long-term follow-up case of adult PM effectively treated with alemtuzumab. Therefore alemtuzumab might be a new, promising alternative in long-term PM therapy. However, randomized and controlled trials are required to draw definite conclusions. Funding: No specific funding was received from any funding bodies in the public, commercial or not-for-profit sectors to carry out the work described in this article. The GTF2I rs117026326 polymorphism is associated with anti-SSA-positive primary Sjö gren's syndrome SIR, Primary SS (pSS) represents a systemic autoimmune disease mainly targeting exocrine glands and manifesting as dryness of the mouth (xerostomia) and eyes (xerophthalmia) [bib_ref] Immunotherapy of myositis: issues, concerns and future prospects, Dalakas [/bib_ref]. It is characterized by lymphocytic infiltrations in these glands and by the presence of a panel of circulating autoantibodies. Among these, those directed against SSA and SSB are the most abundant [bib_ref] Immunotherapy of myositis: issues, concerns and future prospects, Dalakas [/bib_ref]. As is common for autoimmune diseases, the pathogenesis of pSS involves genetic predisposition as well as environmental factors. Determining the genetic basis of pSS will provide an important research tool for exploring the pathogenesis of the disease. In a recent study published in Nature Genetics, Li et al. [2] reported the first genome-wide association study for pSS. In addition to the HLA locus, three non-HLA susceptibility loci, GTF2I, STAT4 and TNFAIP3, were identified in their study . Unexpectedly, the locus indicating the strongest susceptibility was not the HLA locus but the GTF2I rs117026326 C/T polymorphism, with the T allele as the risk allele (OR 2.20, P = 1.31 Â 10 À53 ) for pSS. Moreover, the latter locus has not been reported so far to be associated with any other autoimmune diseases [3, 4], indicating a potential pSS-specific genetic predisposition. The discovery of the GTF2I rs117026326 polymorphism as a strong and disease-specific susceptibility locus provides a hint regarding pSS-specific pathogenesis. Therefore, confirmation of disease relevance and a more detailed analysis of this locus will help considerably in exploring the pathogenesis of pSS. The GTF2I rs117026326 polymorphism was reported to be associated with pSS in Han Chinese but not in Caucasians [2, 5], suggesting an ethnic-specific Letters to the editor [fig] FIG. 1: Histology, clinical disease course and creatine kinase levels in a patient with PM (A) Immunohistochemical staining for CD3 (left) and CD8 (right) showing endomysial inflammatory infiltrates as well as single CD8 + T cells within muscle fibres. (B) Time course of creatine kinase levels, prednisolone dose and maximum walking distance under different treatment regimes. The time period of stable disease after alemtuzumab infusion is highlighted. Letters to the Editor www.rheumatology.oxfordjournals.org [/fig] [table] 5 Taylor: PC, Quattrocchi E, Mallett S et al. [/table]

Tissue fibrocytes in patients with mild asthma: A possible link to thickness of reticular basement membrane? Background: Myofibroblasts, proposed as being derived from circulating fibrocytes, are considered to be important cells in thickening of the basement membrane in patients with asthma. We have studied the correlation of tissue fibrocyte levels to basement membrane thickness and the presence of fibrocytes in bronchoalveolar lavage fluid (BALF) in steroid-naive patients with mild asthma and controls.Methods: Patients with mild asthma (n = 9) were recruited and divided into two categories based on whether or not fibroblast-like cells could be established from BALF. Non-asthmatic healthy subjects (n = 5) were used as controls. Colocalization of the fibrocyte markers CD34, CD45RO, procollagen I, and α-smooth muscle actin (α-SMA) were identified in bronchial biopsies from patients and controls by confocal microscopy. Kruskall-Wallis method was used to calculate statistical significance and Spearman coefficient of rank correlation was used to assess the degree of association. # Background Asthma is characterized by airway inflammation and structural remodeling of the lung [bib_ref] Asthma: mechanisms of disease persistence and progression, Cohn [/bib_ref]. These processes may result in sub-epithelial fibrosis that leads to thickening of the lamina reticularis, which contains an increased amount of connective tissue components such as collagens, tenascin, fibronectin and proteoglycans [bib_ref] Airway remodeling in asthma: new insights, Davies [/bib_ref] [bib_ref] Enhanced proteoglycan deposition in the airway wall of atopic asthmatics, Huang [/bib_ref] [bib_ref] Early thickening of the reticular basement membrane in children with difficult asthma, Payne [/bib_ref]. Fibroblasts are considered to be important key cells in this mechanism since they migrate to sites of injury where they acquire a myofibroblast phenotype, upon activation by factors such as TGF-β1, which is followed by production of a new extracellular matrix [bib_ref] Myofibroblasts and mechano-regulation of connective tissue remodelling, Tomasek [/bib_ref]. However, considering the importance of fibroblasts in airway remodeling it is important to establish the possible origins of these cells which today are unclear. It has been postulated in several in vivo models and in patients with allergen-induced asthma that lung tissue repair and regeneration after injury involve the selective recruitment of circulating fibroblast progenitor cells, termed fibrocytes [bib_ref] Cells derived from the circulation contribute to the repair of lung injury, Abe [/bib_ref] [bib_ref] The peripheral blood fibrocyte is a potent antigen-presenting cell capable of priming..., Chesney [/bib_ref] [bib_ref] Identification of circulating fibrocytes as precursors of bronchial myofibroblasts in asthma, Schmidt [/bib_ref]. These cells comprise a minor component (<1%) of the circulating pool of leukocytes and have been characterized as antigen presenting cells, which express a specific pattern of both mesenchymal and hematopoetic markers including procollagen I, alpha-smooth muscle actin (α-SMA), CXCR4, CD34, and CD45RO [bib_ref] Circulating fibrocytes define a new leukocyte subpopulation that mediates tissue repair, Bucala [/bib_ref]. Fibrocytes have been shown to be important in the generation of fibrosis in several in vivo models where inhibition of fibrocyte recruitment leads to a decrease of fibrotic tissue [bib_ref] CCR2-mediated recruitment of fibrocytes to the alveolar space after fibrotic injury, Moore [/bib_ref] [bib_ref] Fibrocytes contribute to the myofibroblast population in wounded skin and originate from..., Mori [/bib_ref] [bib_ref] Circulating fibrocytes traffic to the lungs in response to CXCL12 and mediate..., Phillips [/bib_ref]. Furthermore, TGF-β1 is an important inducer of fibrosis and may in addition stimulate fibroblasts in tissue and induce the differentiation of fibrocytes into myofibroblast-like cells with elevated production of extracellular matrix components [bib_ref] Peripheral blood fibrocytes: differentiation pathway and migration to wound sites, Abe [/bib_ref] [bib_ref] Altered expression of small proteoglycans, collagen, and transforming growth factor-beta 1 in..., Westergren-Thorsson [/bib_ref]. Although these findings suggest that fibrocytes are important key cells in airway remodeling in patients with allergen-induced asthma and in vivo models, fibrocytes have not been studied in steroid-naive patients with mild asthma. We have previously shown that activated fibroblasts-like cells may be cultured from bronchoalveolar lavage fluid (BALF) from a sub-group of patients with mild asthma accompanied by elevated levels of eosinophils [bib_ref] Presence of activated mobile fibroblasts in bronchoalveolar lavage from patients with mild..., Larsen [/bib_ref]. These BALF fibroblasts could originate from circulating progenitor cells, such as fibrocytes, which may differentiate into myofibroblast-like cells. We hypothesized therefore if fibrocytes were present in remodeled lung tissue and BALF from steroid-naive patients with mild asthma and, if so, correlated with a thickened basement membrane. # Methods ## Subjects, bronchoalveolar lavage and sampling of lung tissue Patients (n = 9) suffering from mild asthma and definite bronchial hyperresponsiveness were included in the study, reviewed and approved by the Swedish Research Ethical Committee (LU339-00). These patients were 25-31 years of age with positive phadiotope staining, nonsmokers, PD 20 < 2 mg/ml of methacholine stimulation, displayed stable mild persistent asthmatic conditions with normal spirometry baseline, free of infections six weeks before bronchoscopy, and had no corticosteroid treatment for six months prior to the study. All of the patients with asthma were atopic and sensitive to pets (cat allergy). Five of the patients with asthma had perennial allergy and one patient had seasonal allergy only (birch pollen). The patients with asthma were further divided into two groups based on whether they displayed the previously described BALF fibroblasts or not (n = 5 and 4, respectively). The control subjects used (n = 5) were 24-51 years of age. These subjects were non-asthmatic healthy volunteers with a reversibility in FEV 1 < 12%, with no allergic symptoms, and who did not respond to doses of methacholine lower than 2 mg/ml. BAL was performed by instillation of buffered saline solution with a recovery of more than 70%. Bronchial biopsies were taken from the central airways of the right lung and collected as previously described [bib_ref] Proteoglycan and proteome profiling of central human pulmonary fibrotic tissue utilizing miniaturized..., Malmstrom [/bib_ref]. Five biopsies were taken from each subject. The biopsies collected did not show any differences in size, vascularity, or muscle content. From each individual 16 sections were analyzed. ## Cell cultures and cytospin analysis Primary fibroblasts-like cells were established from BALF as previously described [bib_ref] Correlation between the bronchial subepithelial layer and whole airway wall thickness in..., Kasahara [/bib_ref]. Briefly, BALF was centrifuged at 500 g for 10 min and the supernatant was discarded and Fibrocyte markers expressed in the BALF fibroblasts. Fibrocyte markers expressed in the BALF fibroblasts. This table displays the number of positive cells in passage 3-6 from BALF fibroblast cultures from patients with mild asthma (n = 5) stained for the different fibrocyte markers used in the study. the precipitated BALF cells were washed with DMEM. The precipitated cells were cultured in DMEM supplemented with 10% Fetal Clone III (Hyclone, UT), 1% L-glutamine, 0.5% gentamicin and 5 µg/ml amfoterracin, until outgrowth of fibroblast-like cells reached confluence (20-30 days). These fibroblast-like cells were characterized by expression of α-SMA, collagen, and high production of proteoglycans [bib_ref] Presence of activated mobile fibroblasts in bronchoalveolar lavage from patients with mild..., Larsen [/bib_ref]. The cultures were trypsinized and used in passage 4-7. Cytospin was performed using a cytocentrifuge (Shandon Southern Products Ltd, Runcorn, Cheshire, UK) where cells in the BALF were collected. The cells were stained using the May-Grünwald/ Giemsa (Sigma Aldrich, Stockholm, Sweden) method according to instructions from the manufacturer. 200 cells were counted and granulocytes were identified according to their morphological structure. ## Histological studies and measurement of basement membrane thickness Bronchial biopsies were fixed in 4% paraformaldehyd, embedded in paraffin and sectioned (5 µm). Basic morphological characterization was made using Gomoris Trichrome staining kit (Polysciences Inc, Warrington, PA) for staining of nuclei, muscles fibers and collagen I. Photographs were taken using a light microscope camera (DM IRB, Leica, Wetzlar, Germany). Basement membrane thickness was quantified by measuring the area of the entire basement membrane, from the base of the bronchial epithelium to the outer limit of the lamina reticularis, and the length of the true basement membrane. This was performed using a computerized image (Leica Software Q500IW, Leica, Wetzlar, Germany). Thickness was calculated as area/length of the basement membrane. These measurements were performed as previously described [bib_ref] New aspects of degranulation and fates of airway mucosal eosinophils, Erjefalt [/bib_ref]. ## Confocal microscopy Embedded and fixed tissue sections (5 µm) and cultured BALF fibroblasts were dehydrated and incubated with primary monoclonal mouse anti-human antibodies: CD34, CD45RO (BD Biosciences, Pharmingen, Leiden, The Netherlands) and α-SMA with Cy3 conjugates (Sigma Aldrich, Stockholm, Sweden). For cultured BALF fibroblast, cells were fixed in 4% paraformaldehyde and 0.5% Triton X-100 in PBS was used as a permeabilization agent prior α-SMA staining. Secondary antibodies used for detection of CD34 and CD45RO were Alexa Fluor 488 and 647 (Molecular Probes, Eugene, OR), respectively. CD45RO and prolyl-4-hydroxylase detection; fixed tissue sections were dehydrated, treated with 1% trypsin, covered with blocking solution (PBS containing 3%BSA, 5% fatfree milkpowder, and 1% goat serum) and incubated with primary monoclonal antibody CD45RO (BD Biosciences, Pharmingen, Leiden, The Netherlands), prolyl-4hydroxylase (Dakocytomation, Denmark) and nucleic staining (Sytox Blue Nucleic Acid Stain, Molecular Probes, Eugene, OR). Sections were then washed with TBS+0.5% Triton X. Secondary antibodies used for detection of CD45RO and prolyl-4-hydroxylase were Alexa Flour 488 and 647 (Molecular Probes, Eugene, OR), respectively. For CD34 and procollagen I detection; fixed tissue sections were dehydrated, treated with 1% trypsin, covered with blocking solution (PBS containing 3%BSA, 5% fatfree milk powder, and 1% goat serum) and incubated with primary antibody mouse anti-human CD34 (BD Biosciences, Pharmingen, Leiden, The Netherlands), rat anti-human procollagen I (Chemicon, Europe Ltd, Hampshire, United Kingdom and nucleic staining (Sytox Blue Nucleic Acid Stain, Molecular Probes, Eugene, OR). Sec- tions were then wash with TBS+0.5% Triton X and blocked with 10% goat serum (Vector Laboratories, Burlingame, CA, USA) in PBS. Secondary antibodies used for detection of CD34 and procollagen I were Alexa Flour 488 and 633 (Molecular Probes, Eugene, OR), respectively. Control experiments were performed with/without primary antibody or with/without secondary antibody. Control sections were included in all experiments to correct for background fluorescence. Isotype controls were used in combination and for each fluorochrome to decrease the risk of detecting unspecific staining. Fibrocytes were counted in a subepithelial zone 0-210 µm deep into the reticular zone and divided into area A, B and C (each area 70 µm deep). Results were expressed as the number of cells per 1 mm 2 . All sections with an intact basement membrane and a minimum requirement of intact tissue 70 µm into the reticular zone were counted. Sections were analyzed with Leica confocal-scanning equipment TCS SP2 II (Leica, Wetzlar, Germany). ## Criteria used when counting fibrocytes in bronchial biopsies ## Quantification of fibrocytes in cultured balf fibroblasts BALF fibroblasts (5000 cells) were cultured 48 h in 4 well chambers. Thereafter 200 cells were randomly choosed for cell counting for their different markers for fibrocytes differentiation. # Statistical analysis Mean values ± standard errors of the mean (SEM) were calculated and Kruskall-Wallis was used for analyses of statistical significance (SPSS v7.0, Chicago, IL). All values of p < 0.05 were considered significant. Spearman coefficient of rank correlation was used to assess the degree of association between basement membrane thickness and number of fibrocytes. # Results ## Tissue fibrocytes are localized in proximity to the basement membrane in patients with mild asthma The sub-epithelial bronchial tissues were divided into three sections (A, B, and C) when studying the localization of fibrocytes [fig_ref] Figure 1: Criteria used when counting fibrocytes in bronchial biopsies [/fig_ref]. The presence of double stained cells could be analyzed by using antibodies for CD34/α-SMA , CD45RO/α-SMA , CD34/CD45RO , and analyzed by confocal microscope. The indication of a fibrocytes phenotype could be demonstrated by colocalization of all these three markers . Fibrocytes were primarily localized individually and in clusters close to the epithelium and to blood vessels . Limited background staining was seen throughout the confocal analysis, indicating specificity for the antibodies used . Single positive cells were also detected close to triple positive cells in the basement membrane proximity. Apart from expressing CD34/CD45RO, fibrocytes are further characterized as being collagen producing cells. The cells were therefore stained for prolyl-4-hydroxylase, a protein critically involved in collagen synthesis, and procollagen I. Prolyl-4-hydroxylase/CD45RO could be seen in separate cells and in clusters of cells . These two markers were also colocalized as shown by a merged picture. The cells did also stain procollagen I/CD34 and the merged picture indicate coexpression of these markers . Collectively, the merged pictures illustrate positive cells for these markers suggesting an active collagen production in these fibrocytes. When counting number of fibrocytes in the tissue, patients with BALF fibroblasts displayed a 14-fold increase (p < 0.01) in the total number of triple positive CD34/CD45RO/α-SMA fibrocytes present in area A + B when compared to controls . The other patient group without fibroblasts in BALF displayed a significant 2-fold increase (p < 0.05) in fibrocytes when compared to controls. The localization of triple positive cells strongly suggests a gradient of fibrocytes present throughout these areas. In area A, patients with BALF fibroblasts displayed an 11-fold increase of fibrocytes (p < 0.05) when compared to controls (52 ± 32 vs. 5 ± 2) and a 5-fold increase when compared to patients without BALF fibroblasts (52 ± 32 vs. 12 ± 1). This pattern was similar in area B where fibrocytes were observed in patients with and without BALF fibroblasts (11 ± 6 vs. 4 ± 2) but not in the controls. In area C, fibrocytes were only detected in patients with BALF fibroblasts (3 ± 1) and controls (1 ± 1). When quantifying the number of prolyl-4-hydroxylase/CD45RO positive cells, patients with BALF fibroblasts showed 2.5-fold elevated levels of these cells when compared to the other patient group (p < 0.05) and 17-fold increase when compared to controls (p < 0.05) . A similar pattern was seen when quantifying CD34/procollagen I positive cells , where a 2-fold increase (p < 0.05) in double positive cells were seen in patients with BALF fibroblasts when compared to the other patient group and a 16-fold increase when compared to controls (p < 0.05). ## Fibrocytes levels correlate to thickness of basement membrane The basement membrane thickness was measured in controls , patients with asthma with BALF fibroblasts , and patients without BALF fibroblasts . The basement membrane was significantly thicker in patients with asthma with BALF fibroblast (7.95 ± 0.98 µm) when compared to the other patient group and controls (6.12 ± 0.18 µm and 5.23 ± 0.4 µm respectively, p < 0.05) . A higher number of cells detected by hematoxylin staining beneath the basement membrane when compared to controls were characteristic for both groups of patients. Moreover, our data indicate a correlation between thickness of basement membrane and the presence of fibrocytes in the tissue of controls and patients with asthma (r = 0.711) . ## Balf fibroblasts display fibrocyte characteristics BALF fibroblasts displayed an elongated, spindle-like shape after 20-30 days in culture . So far, the origin of cultured BALF fibroblasts from patients with mild asthma has not been studied in detail. To see whether the BALF fibroblasts displayed characteristics of Fibrocytes in patients with mild asthma express collagen Fibrocytes in patients with mild asthma express collagen. Bronchial biopsies were stained using immunofluorescent antibodies against prolyl-4-hydroxylase (green) CD45RO (red) and nuclei (blue) (A). Squares indicate the same cluster area as shown in A. The merged picture describes the positive cells within the square. Cells were further stained for CD34 (green), procollagen I (red), and nuclei (blue) (B), and were subjected to confocal microscopy. The merged picture describes the positive cell within the square. Definition of abbreviations: BM = Basement membrane fibrocytes, these cells were triple-stained for CD34, CD45RO, and α-SMA, and subjected to confocal microscopy. Again, insignificant levels of background staining were seen indicating specificity for the antibodies used (data not shown). The BALF fibroblasts did indeed express the fibrocyte markers CD34, CD45RO, and α-SMA, which suggest that these cells may be derived from fibrocytes . The number of BALF fibroblasts in passage 3-6 expressing all three fibrocyte markers constituted one fifth of the total cell count. Double-positive cells expressing CD34/α-SMA or CD45RO/α-SMA constituted 11.7 % and 5% respectively [fig_ref] Figure 1: Criteria used when counting fibrocytes in bronchial biopsies [/fig_ref]. Moreover, 41 % of the cells stained positive only for α-SMA. Inflammatory cells such as eosinophils are suggested to be important in mediating the airway inflammation in patients with asthma and in vivo models [bib_ref] Asthma: mechanisms of disease persistence and progression, Cohn [/bib_ref] [bib_ref] Remodeling in asthma and chronic obstructive lung disease, Jeffery [/bib_ref]. Increased levels of BALF eosinophils have been reported in patients with BALF fibroblasts, which suggests an active inflammatory link to the presence of these fibroblasts [bib_ref] Airway inflammation, basement membrane thickening and bronchial hyperresponsiveness in asthma, Ward [/bib_ref]. However, we did not observe any correlation between the number of BALF eosinophils and fibrocytes in lung tissue from patients with mild asthma in this study (data not shown). # Discussion This is the first study to report an increased number of fibrocytes expressing CD34/CD45RO/α-SMA/procollagen I, and a localization of these cells to areas close to the basement membrane in steroid-naive patients with mild asthma when compared to controls. The inclusion of α-SMA coexpression to the other fibrocyte markers may indicate a possible link between circulating progenitor cells and their potential differentiation into α-SMA expressing myofibroblasts in these patients. It should be noted that the patients included in this study is very homogenous since they did not show any distinct clinical patterns except for five patients that had perennial allergy and were sensitive to cat. Fibrocyte levels correlated to the thickness of the basement membrane proposing that these cells may participate in airway wall remodeling. The relative high levels of fibrocytes expressing α-SMA seen in patients with increased basement membrane thickness may indicate a more differentiated fibrocyte phenotype that is similar to the myofibroblast. In contrast to our findings, Schmidt and coworkers reported higher levels of collagen I expressing cells than α-SMA positive cells in ovalbumin treated mice and patients with allergen-provoked asthma [bib_ref] Myofibroblast involvement in the allergen-induced late response in mild atopic asthma, Gizycki [/bib_ref]. Apart from the different study models used, these differences may be caused by alteration in cell differentiation which is difficult to monitor for fibrocytes in particular. Importantly, the analysis of prolyl-4-hydroxylase in our study indicates an active collagen production in the reported fibrocytes. To our knowledge, this is the first study to report a correlation between tissue fibrocytes and increased thickness of basement membrane. Thickening of the lamina reticularis has been reported in the airways of children with severe asthma and has been shown to correlate to airway hyperresponsiveness, as well as an increased number of fibroblasts and myofibroblast in this area [bib_ref] Induction of epithelial-mesenchymal transition in alveolar epithelial cells by transforming growth factor-beta1:..., Willis [/bib_ref] [bib_ref] Progenitor cell trafficking is regulated by hypoxic gradients through HIF-1 induction of..., Ceradini [/bib_ref] [bib_ref] Bone marrowderived progenitor cells in pulmonary fibrosis, Hashimoto [/bib_ref]. Apart from Quantification of CD34, CD45RO, α-SMA, procollagen I and prolyl-4-hydroxylase coexpression in fibrocytes Quantification of CD34, CD45RO, α-SMA, procollagen I and prolyl-4-hydroxylase coexpression in fibrocytes. Cells in bronchial biopsies coexpressing CD34/CD45RO/α-SMA (A), CD45RO/prolyl-4-hydroxylase (B), and CD34/procollagen I (C), were counted. These cells were counted in the areas A and B and data included are from controls, patient with mild asthma with (+) and without (-) BALF fibroblasts and are presented as positive cells/mm 2 of tissue section. Values are presented as means ± SEM, n = 4-5 patients. the differentiation of fibrocytes into myofibroblasts-like cells, additional sources of myofibroblasts, that may contribute to the enhanced extracellular matrix deposition during pathological conditions include resident myofibroblasts, differentiation of bronchial muscle cells and epithelial mesenchymal transition [bib_ref] G-CSF induces stem cell mobilization by decreasing bone marrow SDF-1 and up-regulating..., Petit [/bib_ref]. Molecules secreted by airway epithelium and surrounding inflammatory cells may explain the observed difference in numbers of fibrocytes in area A compared to area B and C. Factors that have previously been shown to be involved in the recruitment of fibrocytes to the fibrotic tissue include IL-1β, ED-A fibronectin, stromal-derived factor 1 (SDF1/ CXCL12), granulocyte macrophage stimulating-factor (GM-CSF). Hence, the differentiated levels of fibrocytes and basement membrane thickness seen in this study may be a result of an altered local environment in the lungs of the two sub-groups of patients with mild asthma. Future screening of fibrocyte recruiting molecules in the BALF from these patients could provide a better understanding of the differences observed. The outgrowth of fibroblasts in BALF from a sub-group of patients with mild asthma indicate a possible origin from circulating fibroblast progenitor cells, due to their relative high expression of fibrocyte markers CD34, CD45RO and α-SMA. Since the airway lumen has low levels of extracellular matrix, which is a prerequisite for survival of mature fibroblast, it is very likely that the BALF fibroblasts are derived from circulating fibrocytes rather than the residing tissue fibroblast pool. These lumen-derived cells were seen to correlate to the number of tissue fibrocytes and basement membrane thickness, indicating a possible link to the pathological changes in these patients. When human fibrocytes are cultured in vitro, a decrease in the expression of CD34 and CD45RO occurs in parallel with an increased expression of α-SMA. This may confirm why one third of the original fibrocytes stained positive for α-SMA after three weeks. Similarly, approximately 50 % of the cells were stained positive only for α-SMA in passage 3-6 . In the same passages, triple positive cells constituted approximately a fifth of the total cell count and double-positive for CD34/α-SMA or CD45RO/ α-SMA constituted 10 % and 5 % respectively. This allow us to speculate that a homogenous population of CD34/ CD45RO expressing fibrocytes in the BALF may have been the starting population, which has gradually differentiated into α-SMA expressing myofibroblast-like cells. Future studies, including detailed analysis of recovered cells expressing fibrocyte specific markers in BALF, may provide a clearer future view into this field. BALF fibroblasts have previously been characterized as motile cells with a relatively high production of extracellular matrix components, including collagen and proteoglycans, accompanied by elevated levels of BALF eosinophils. In the present study, we did not observe any correlation of neither BALF nor tissue eosinophils to the number of fibrocytes in tissue. A possible explanation is that BALF eosinophils affect the differentiation of fibrocytes into myofibroblast-like cells due to their capacity of secreting TGF-β, rather than the recruitment of these cells to the lumen. In addition, we have preliminary data indicating a correlation between BALF neutrophils and tissue fibrocytes, suggesting an alternative inflammatory link to the recruitment of fibrocytes to the tissue (unpublished observation). Basement membrane thickness in patients with mild asthma and controls Basement membrane thickness in patients with mild asthma and controls. Bronchial biopsies from controls (n = 5) (A), patients with mild asthma with (+) BALF fibroblasts (n = 5) (B) and patients with mild asthma without (-) BALF fibroblasts (n = 4) (C) were stained with Gomoris trichrome staining for basic morphological characterization. Measurement of basement membrane thickness was performed as described in the Method section and is presented in µm (D). Number of fibrocytes in area A+B were counted and correlated (r = 0.711) to thickness of basement membrane (µm) (E). Values are presented as means ± SEM. Spearman coefficient of rank correlation was used to calculate the degree of association between basement membrane thickness and number of fibrocytes. (▲ patients with BALF fibroblasts, ▪ patients without BALF fibroblasts, - controls). Definition of abbreviations: BM = Basement membrane, E = epithelium, MF = Muscle fibers. An important question remains to be answered: Do fibrocytes differentiate into myofibroblasts, or do they stimulate differentiation and proliferation of other progenitor cells or resident mesenchymal cells within the tissue? In conclusion, the novel findings that cells from BALF in patients with mild asthma express fibrocyte specific proteins and the localization in tissue of these cells in close proximity to a thicker basement membrane emphasize the importance of these cells in the early stages of remodeling and inflammation. Further exploration in patients with severe asthma would be of paramount interest. The observation that patients with BALF fibroblasts with fibrocyte characteristics are accompanied by a thicker basement membrane suggests a specific pathological process for these patients when compared to patients lacking these cells. In the future, modulation of the recruitment and subsequent differentiation of fibrocytes may therefore be of great therapeutic importance in regulating airway wall remodeling in asthma. # Conclusion In conclusion, we hereby show that cells from BALF in patients with mild asthma express fibrocyte specific proteins and that these are localized in tissue in close proximity to a thicker basement membrane. These data, together with recent fibrocyte findings, emphasize the importance of these cells in airway remodeling in patients with mild asthma. BALF fibroblasts in patients with mild asthma express the fibrocyte markers CD34, CD45RO, and α-SMA BALF fibroblasts in patients with mild asthma express the fibrocyte markers CD34, CD45RO, and α-SMA. BALF fibroblasts were cultured as described in the method section and observed after approximately 30 days in culture (A). The BALF fibroblasts were fixed in 4% paraformaldehyde and stained with antibodies against CD34 (green), CD45RO (red) and α-SMA (blue) (B). These pictures were merged together with a transmission picture. [fig] Figure 1: Criteria used when counting fibrocytes in bronchial biopsies. Sections from patients and controls were based on an intact basement membrane together with intact tissue of at least 70 µm in every section. Area A starts were the basement membrane ends. A = 0-70 µm in the section. B = 71-140 µm in the section and C = 141-210 µm in the tissue. Definition of abbreviations: BM = Basement membrane, E = epithelium, MF = Muscle fibers. [/fig]

Role of Complement in Multiorgan Failure Multiorgan failure (MOF) represents the leading cause of death in patients with sepsis and systemic inflammatory response syndrome (SIRS) following severe trauma. The underlying immune response is highly complex and involves activation of the complement system as a crucial entity of innate immunity. Uncontrolled activation of the complement system during sepsis and SIRS with in excessive generation of complement activation products contributes to an ensuing dysfunction of various organ systems. In the present review, mechanisms of the inflammatory response in the development of MOF in sepsis and SIRS with particular focus on the complement system are discussed. # Introduction In the 1970s, a syndrome of progressive, sequentially dysfunctional organ systems has been firstly characterized, eventually referred to as multiorgan failure (MOF) [bib_ref] Multiple, progressive, or sequential systems failure. A syndrome of the 1970s, Baue [/bib_ref] [bib_ref] Causes of death and determinants of outcome in critically ill patients, Mayr [/bib_ref]. As a predominant underlying condition, sepsis and sepsisassociated MOF represent one of the leading causes of death of hospitalized patients with reported morality rates ranging from 28% to 56% [bib_ref] Incidence, organ dysfunction and mortality in severe sepsis: a Spanish multicentre study, Blanco [/bib_ref] [bib_ref] The epidemiology of sepsis in the United States from, Martin [/bib_ref]. Likewise, severe trauma and trauma-related multiorgan failure remain the leading cause of death in people below the age of 40 [bib_ref] Trauma fatalities: time and location of hospital deaths, Demetriades [/bib_ref] [bib_ref] Epidemiology of trauma deaths: a reassessment, Sauaia [/bib_ref]. The conception of organ failure has changed over the years and various scoring systems for the classification and diagnosis of MOF exist all of which attempt to quantify the degree of organ failure [bib_ref] MOF, MODS, and SIRS: what is in a name or an acronym, Baue [/bib_ref] [bib_ref] Multiple organ dysfunction score: a reliable descriptor of a complex clinical outcome, Marshall [/bib_ref] [bib_ref] The SOFA (Sepsisrelated Organ Failure Assessment) score to describe organ dysfunction/failure, Vincent [/bib_ref]. Currently, MOF is regarded as a continuous process of varying levels of organ failure rather than an all-or-none event [bib_ref] Multiple Organ Failure in Trauma Patients, Durham [/bib_ref]. To characterize MOF, six different organ systems are regarded as "key organs": lungs, cardiovascular system, kidneys, liver, coagulation system, and central nervous system. Depending on the severity and various predisposing conditions, the initial insult (tissue trauma, infection) can induce a systemic host response that is characterized by the release of pro-and anti-inflammatory cytokines and meta-bolites (e.g., reactive oxygen (ROS) and nitrogen species (NOS)), activation of plasmatic cascade systems, such as the complement and the coagulation systems, and the appearance of acute phase proteins as well as hormonal and neuronal mediators [bib_ref] Pathophysiology of polytrauma, Keel [/bib_ref] [bib_ref] The pathophysiology and treatment of sepsis, Hotchkiss [/bib_ref] [bib_ref] Harmful molecular mechanisms in sepsis, Rittirsch [/bib_ref]. Imbalanced systemic immune responses can ultimately lead to accumulation of leukocytes, disseminated intravascular coagulation (DIC), and microcirculatory dysfunction with subsequent apoptosis and necrosis of parenchymal cells, finally resulting in the development of MOF [bib_ref] The pathophysiology and treatment of sepsis, Hotchkiss [/bib_ref] [bib_ref] Apoptotic cell death in patients with sepsis, shock, and multiple organ dysfunction, Hotchkiss [/bib_ref] [bib_ref] Immunologic assessment of host defense impairment in patients with septic multiple organ..., Goya [/bib_ref]. As a central entity of innate immunity, the complement system is immediately activated after trauma or infection in order to control the replication of intruding pathogens. In humans, the plasma levels of complement activation products rise early, are persistently elevated in patients after thermal injury, trauma, and sepsis, and correlate with the severity of injury and inversely with the outcome [bib_ref] Circulating complement proteins in multiple trauma patients-correlation with injury severity, development of..., Hecke [/bib_ref] [bib_ref] Complement activation in injured patients occurs immediately and is dependent on the..., Fosse [/bib_ref] [bib_ref] Change of complement system predicts the outcome of patients with severe thermal..., Kang [/bib_ref] [bib_ref] Altered anaphylatoxin activity during induced hypoperfusion in acute and elective abdominal aortic..., Bengtson [/bib_ref] [bib_ref] Chronological changes in the complement system in sepsis, Nakae [/bib_ref] [bib_ref] Role of the alternative pathway in the early complement activation following major..., Ganter [/bib_ref] [bib_ref] Early complementopathy after multiple injuries in humans, Burk [/bib_ref]. It is well established that activation of the complement cascade alters functional responses of neutrophils (PMN) in the course of systemic inflammation and contributes to the development of organ failure [bib_ref] Immunologic assessment of host defense impairment in patients with septic multiple organ..., Goya [/bib_ref] [bib_ref] The role of the complement system in the pathogenesis of multiple organ..., Zimmermann [/bib_ref]. In experimental sepsis, the blockade of complement anaphylatoxin C5a virtually prevented the appearance of MOF and improved the outcome [bib_ref] Role of C5a in multiorgan failure during sepsis, Huber-Lang [/bib_ref] [bib_ref] Protective effects of C5a blockade in sepsis, Czermak [/bib_ref] [bib_ref] Functional roles for C5a receptors in sepsis, Rittirsch [/bib_ref]. Previous studies strongly suggest a mutual crosstalk between the complement and the coagulation system [bib_ref] Anti-C5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis, Laudes [/bib_ref] [bib_ref] Generation of C5a in the absence of C3: a new complement activation..., Huber-Lang [/bib_ref] [bib_ref] Complement and coagulation: strangers or partners in crime?, Markiewski [/bib_ref] [bib_ref] Interaction between the coagulation and complement system, Amara [/bib_ref]. Due to the complex nature of plasmatic cascades and their interconnections, the role and regulations of the complement system, especially in states of disease, are still inadequately understood. This article is sought to provide insights into the pathogenesis of multiorgan failure associated with systemic inflammation with particular focus on the role of the complement system. Furthermore, potential therapeutic strategies targeting the complement cascade to prevent the development of MOF as well as possible future research directions are addressed. ## Pathways of complement activation The complement system can be activated via four different pathways, the classical, the alternative, and the lectin pathway [bib_ref] The proteolytic activation systems of complement, Reid [/bib_ref] [bib_ref] Molecular organization and function of the complement system, Muller-Eberhard [/bib_ref] [bib_ref] Evolution of the lectin-complement pathway and its role in innate immunity, Fujita [/bib_ref]. All three pathways lead to the assembly of the C3 convertase which cleaves C3 into C3a and C3b [bib_ref] The proteolytic activation systems of complement, Reid [/bib_ref] [bib_ref] Molecular organization and function of the complement system, Muller-Eberhard [/bib_ref]. Incorporation of C3b into the C3 convertase results in formation of the C5 convertase, which cleaves C5 into C5a and C5b. The split products C3a and C5a act as potent anaphylatoxins. C3b is an important opsonic factor, while C5b initiates the formation the membrane attack complex (C5b-9). In addition, various non-complement serine proteases seem to cleave complement components into biologically active complement products with variable efficacy [bib_ref] Molecular intercommunication between the complement and coagulation systems, Amara [/bib_ref]. In particular, thrombin has been found to function as a C5-convertase that does not require the presence of C3 or C3b [bib_ref] Generation of C5a in the absence of C3: a new complement activation..., Huber-Lang [/bib_ref]. Moreover, proteases from PMN and macrophages can cleave C5 as well [bib_ref] Generation of C5a by phagocytic cells, Huber-Lang [/bib_ref] [bib_ref] Cleavage of the fifth component of complement and generation of a functionally..., Vogt [/bib_ref]. There is evidence that all three complement activation pathways are activated in SIRS and sepsis. Interestingly, it has been demonstrated that during the course of sepsis alternative pathway activation occurs earlier than activation of the classical pathway [bib_ref] Complement activation in septic shock patients, Sprung [/bib_ref]. Based on their distinct mechanisms and kinetics of activation, it has been hypothesized that classical pathway activation in sepsis plays a crucial role in the clearance of pathogenic factors, while the alternative pathway is thought to be essential for fighting against infections by invading microorganisms [bib_ref] The role of complement system in septic shock, Charchaflieh [/bib_ref]. Although the knowledge about the underlying mechanisms is limited, recent reports suggest a particular role of mannose-binding lectin (MBL) and the lectin pathway in the development of MOF. In sterile systemic inflammation (systemic inflammatory response syndrome, SIRS), patients with functional MBL deficiency due to MBL consumption did not develop MOF unless MBL was reconstituted by transfusion of fresh frozen plasma [bib_ref] Mannose-binding lectin is involved in multiple organ dysfunction syndrome after cardiac surgery:..., Bilgin [/bib_ref]. In contrast, septic patients with MBL depletion showed significantly higher sequential organ failure assessment (SOFA) scores, whereas functional MBL levels and activity in sepsis were associated with moderate SOFA scores and better prognosis [bib_ref] Low mannosebinding lectin function is associated with sepsis in adult patients, Eisen [/bib_ref] , suggesting that MBL might be essential for defence against infections on the one hand, but might also harm the host and contribute to the development of MOF on the other hand. Therefore, as indicated by this dual function of the lectin pathway, the role of the complement system in systemic inflammation sometimes is referred to as a doubleedged sword. ## Dysfunction of the central nervous system Historically, the central nervous system (CNS) was defined as an "immunological privileged organ" because of its separation from peripheral circulation by the blood-brain barrier (BBB). However, it became evident that the CNS is a rich source of inflammatory mediators and complement proteins can be produced by neurons, astrocytes, microglia, and oligodendroglia [bib_ref] Expression of complement in the brain: role in health and disease, Morgan [/bib_ref] [bib_ref] Primary cultures of murine astrocytes produce C3 and factor B, two components..., Levi-Strauss [/bib_ref] [bib_ref] Expression of complement messenger RNAs and proteins by human oligodendroglial cells, Hosokawa [/bib_ref]. Severe trauma and sepsis are associated with systemic inflammation that can lead to blood-brain barrier (BBB) dysfunction and cerebral edema, regardless of the presence of traumatic brain injury (TBI) [bib_ref] Closed head injury-an inflammatory disease?, Schmidt [/bib_ref]. The breakdown of the BBB is considered to be a key event in the development of septic encephalopathy, while the cellular and molecular mechanisms of sepsis-induced brain damage are still vastly unknown [bib_ref] Pathophysiology of septic encephalopathy-an unsolved puzzle, Flierl [/bib_ref]. Interestingly, the direct contact between blood and cerebrospinal fluid leads to complement activation, and the extent of intrathecal complement activation is associated with BBB dysfunction [bib_ref] Complement activation in the central nervous system following bloodbrain barrier damage in..., Lindsberg [/bib_ref]. In addition, intracerebral complement levels increase under pathological conditions due to leakage of serumderived complement proteins into the subarachnoidal space after breach of the BBB as well as increased complement biosynthesis in the CNS [bib_ref] The role of the complement system in traumatic brain injury, Stahel [/bib_ref]. C1q, C3a, and C5a contribute to intracranial inflammation by induction of BBB damage and increase in vascular permeability [bib_ref] The role of the complement system in traumatic brain injury, Stahel [/bib_ref] [bib_ref] Microglial activation and increased synthesis of complement component C1q precedes blood-brain barrier..., Lynch [/bib_ref]. Bloodderived leukocytes, predominantly PMN, are then able to transmigrate into the CNS and release proteases and free radicals resulting in tissue damage [fig_ref] Figure 1: Summarizing illustration on the effects of excessive complement activation on various organ... [/fig_ref] [bib_ref] The role of the complement system in traumatic brain injury, Stahel [/bib_ref] [bib_ref] Tissue destruction by neutrophils, Weiss [/bib_ref]. In line with this, in experimental sepsis blockade of C5a attenuated pathophysiological changes that are typically associated with septic encephalopathy [bib_ref] Inhibition of complement C5a prevents breakdown of the blood-brain barrier and pituitary..., Flierl [/bib_ref]. C3 and its derivates seem to play a central role in the pathogenesis of CNS dysfunction. Accumulation of C3 fragments is related to neuronal cell death and intracerebral PMN infiltration [bib_ref] Local neutrophil influx following lateral fluid-percussion brain injury in rats is associated..., Keeling [/bib_ref]. Previous studies suggested that the alternative pathway activation is a leading mechanism for neuronal cell death after closed head injury [bib_ref] Reduced neuronal cell death after experimental brain injury in mice lacking a..., Leinhase [/bib_ref] [bib_ref] Inhibition of the alternative complement activation pathway in traumatic brain injury by..., Leinhase [/bib_ref]. C5a can induce neuronal apoptosis via the interaction with its receptor (C5aR), which is abundantly expressed on various cell types in the CNS [bib_ref] A neuronal C5a receptor and an associated apoptotic signal transduction pathway, Farkas [/bib_ref] [bib_ref] Complement anaphylatoxin receptors on neurons: new tricks for old receptors?, Nataf [/bib_ref]. Finally, inactivation of the complement regulatory proteins on neurons during inflammation pave the road for complement-mediated lysis of homologous cells by the membrane attack complex [bib_ref] Spontaneous classical pathway activation and deficiency of membrane regulators render human neurons..., Singhrao [/bib_ref]. Despite the unambiguous involvement in various pathological mechanisms, the role of the complement system in the pathogenesis of CNS dysfunction appears to be a double-edged sword since it has been reported that C3a as well as C5a also may mediate neuroprotective and neuroregenerative effects [bib_ref] Complement-derived anaphylatoxin C5a protects against 8 Clinical and Developmental Immunology glutamate-mediated neurotoxicity, Osaka [/bib_ref] [bib_ref] Inflammatory signals induce neurotrophin expression in human microglial cells, Heese [/bib_ref]. ## Respiratory failure Respiratory failure or acute respiratory distress syndrome (ARDS) represents a frequent complication after burn injury, multisystem trauma, shock, and systemic inflammation [bib_ref] Role of pulmonary alveolar macrophage activation in acute lung injury after burns..., Clark [/bib_ref] [bib_ref] The epidemiology of posttraumatic adult respiratory distress syndrome, White [/bib_ref] [bib_ref] Incidence and outcomes of acute lung injury, Rubenfeld [/bib_ref]. Although the liver represents the main source for the production of complement proteins, virtually all complement proteins can be locally produced in the lung by type II alveolar pneumocytes, alveolar macrophages, and lung fibroblasts [bib_ref] Human alveolar macrophages synthesize the functional alternative pathway of complement and active..., Hetland [/bib_ref] [bib_ref] Pulmonary alveolar type II epithelial cells synthesize and secrete proteins of the..., Strunk [/bib_ref] [bib_ref] Effect of lipopolysaccharide on C3 and C5 production by human lung cells, Rothman [/bib_ref]. While the total pulmonary complement protein concentration is at comparable levels as found in serum, its activity in normal lung is markedly reduced which is attributed to the ability of surfactant protein A (SPA) to inhibit complement [bib_ref] Complementmediated host defense in the lung, Watford [/bib_ref] [bib_ref] Surfactant protein A regulates complement activation, Watford [/bib_ref]. In various studies, patients with ARDS showed evidence for robust complement activation, the extent of which correlated with the degree and outcome of ARDS [bib_ref] Association of complement activation and elevated plasma-C5a with adult respiratory distress syndrome...., Hammerschmidt [/bib_ref] [bib_ref] Complement activation and clearance in acute illness and injury: evidence for C5a..., Solomkin [/bib_ref]. In particular, the complement anaphylatoxin C5a and the MAC are in the focus of ARDS pathophysiology, but also elevated levels of C3a and C4a have been linked to the development of ARDS [bib_ref] Complement activation and clearance in acute illness and injury: evidence for C5a..., Solomkin [/bib_ref] [bib_ref] Accentuated formation of the terminal C5b-9 complement complex in patient plasma precedes..., Langlois [/bib_ref] [bib_ref] Complement activation and the prognostic value of C3a in patients at risk..., Zilow [/bib_ref] [bib_ref] Biologically active products of complement and acute lung injury in patients with..., Weinberg [/bib_ref] [bib_ref] Role of C3, C5 and anaphylatoxin receptors in acute lung injury and..., Bosmann [/bib_ref]. C5a promotes inflammation by causing extensive influx of activated PMN into lung tissue and the alveolar space and by enhancement of the early cytokine response (reviewed in [bib_ref] Role of C3, C5 and anaphylatoxin receptors in acute lung injury and..., Bosmann [/bib_ref] [bib_ref] Role of C5a in inflammatory responses, Guo [/bib_ref]. However, only little is known about the local regulation of complement activation. Besides, the complement inhibitory function of SPA, C1 inhibitor, which inhibits classical pathway activation, has been detected in human bronchoalveolar lavage fluids [bib_ref] Complementmediated host defense in the lung, Watford [/bib_ref] [bib_ref] Surfactant protein A regulates complement activation, Watford [/bib_ref]. Lung activity of both, surfactant protein and C1 inhibitor, is significantly reduced in patients with trauma-related ARDS [bib_ref] Alveolar surfactant and adult respiratory distress syndrome. Pathogenetic role and therapeutic prospects, Seeger [/bib_ref] [bib_ref] Activation of the contact system of plasma proteolysis in the adult respiratory..., Carvalho [/bib_ref]. Beside complement activation, ARDS is accompanied by tissue factor generation and widespread pulmonary fibrin deposition [bib_ref] Time course of procoagulant activity and D dimer in bronchoalveolar fluid of..., Fuchs-Buder [/bib_ref] [bib_ref] Procoagulant activity in bronchoalveolar lavage of severely traumatized patientsrelation to the development..., Seeger [/bib_ref]. Here, antithrombin III (ATIII), which inhibits activated proteases including thrombin, seems to play a central role since ATIII levels inversely correlate with the outcome in the setting of sepsis, and ATIII has been shown to block the thrombin pathway of complement activation in a murine model of acute lung injury [fig_ref] Figure 1: Summarizing illustration on the effects of excessive complement activation on various organ... [/fig_ref] [bib_ref] Generation of C5a in the absence of C3: a new complement activation..., Huber-Lang [/bib_ref] [bib_ref] Therapeutic use of antithrombin concentrate in sepsis, Balk [/bib_ref] [bib_ref] Coagulation abnormalities in acute lung injury and sepsis, Abraham [/bib_ref]. In conclusion, systemic inflammation provokes local imbalances of the complement and the coagulation cascade shifting the lung equilibrium to a proinflammatory and procoagulant state, which then stimulates accumulated leukocytes to locally release cytokines, enzymes, and radicals that promote the classical features of ARDS. ## Cardiac dysfunction Heart dysfunction during inflammatory states shows a biphasic process with an early hyperdynamic phase followed by a pivotal hypodynamic phase [bib_ref] Myocardial dysfunction in the patient with sepsis, Krishnagopalan [/bib_ref]. Hallmarks of the hypodynamic phase are decreased cardiac output, reduced microvascular flow, and increased peripheral vascular resistance with rising plasma levels of catecholamines. It has been suggested that these changes initiate the vicious circle of multiorgan failure due to compromised organ perfusion, decreased oxygen and nutrient supply, and ischemia [bib_ref] Role of nitric oxide and cGMP in human septic serum-induced depression of..., Kumar [/bib_ref]. Various myocardial depressant factors that collectively trigger cardiac contractility deficits in systemic inflammation have been described, but no single agent responsible for myocardial dysfunction could be identified [bib_ref] Role of nitric oxide and cGMP in human septic serum-induced depression of..., Kumar [/bib_ref] [bib_ref] Leukotrienes C4, D4 and E4: effects on human and guinea-pig cardiac preparations..., Burke [/bib_ref] [bib_ref] Indomethacin suppresses the early cardiodepressant factor released by endotoxin in the rat:..., Carli [/bib_ref] [bib_ref] Isolation of a shock-induced circulating cardiodepressant substance, Goldfarb [/bib_ref] [bib_ref] Tumor necrosis factor-α as a myocardial depressant substance, Odeh [/bib_ref] [bib_ref] A circulating myocardial depressant substance in humans with septic shock. Septic shock..., Parrillo [/bib_ref] [bib_ref] Anaphylotoxin levels following thermal injury, Moran [/bib_ref]. In previous reports, complement activation has been linked to hemodynamic depression, but the mechanisms by which complement activation products might cause dysfunction of cardiomyocytes remain to be defined in detail [bib_ref] Role of nitric oxide and cGMP in human septic serum-induced depression of..., Kumar [/bib_ref] [bib_ref] Systemic complement activation produces hemodynamic changes characteristic of sepsis, Schirmer [/bib_ref] [bib_ref] Complement-mediated hemodynamic depression in the early postburn period, Schirmer [/bib_ref]. In experimental studies, C5a has been demonstrated to induce cardiac dysfunction with impaired cardiomyocyte contractility, which could be restored by blockade of C5a [bib_ref] An essential role for complement C5a in the pathogenesis of septic cardiac..., Niederbichler [/bib_ref] [bib_ref] C5a-blockade improves burn-induced cardiac dysfunction, Hoesel [/bib_ref]. But it is far from certain if C5a-C5aR interaction directly causes cellular alterations in cardiomyocytes that lead to impaired calcium handling, oxygen and ATP depletion, and loss of mitochondria with energy deficit [bib_ref] Calcium uptake by sarcoplasmic reticulum is impaired during the hypodynamic phase of..., Wu [/bib_ref] [bib_ref] Metabolic dysfunction and depletion of mitochondria in hearts of septic rats, Watts [/bib_ref]. Recent research suggests that C5a causes the local release of cardiosuppressive cytokines and chemokines in cardiomyocytes eventually leading to cardiac dysfunction [bib_ref] Complement dependency of cardiomyocyte release of mediators during sepsis, Atefi [/bib_ref]. But it is also conceivable that complement anaphylatoxins contribute to induce "hibernation" in cardiomyocytes as it occurs in the response of the myocardium to ischemia [bib_ref] Evidence of myocardial hibernation in the septic heart, Levy [/bib_ref]. In the ischemic heart, it is a common observation that the induction of contractile dysfunction by C5a is not a direct effect but rather involves secondary production of mediators (e.g., arachidonic acid metabolites), which then act on target cells [fig_ref] Figure 1: Summarizing illustration on the effects of excessive complement activation on various organ... [/fig_ref] [bib_ref] Complement activation in heart diseases: role of oxidants, Chakraborti [/bib_ref]. Further, predominantly the classical and the alternative pathway are activated upon myocardial ischemia. Treatment with C1 inhibitor or soluble complement receptor 1 has cardioprotective effects by suppression of adhesion molecule expression (p-selectin, ICAM-1), blockade of C3 deposition and its activity on cardiomyocytes, and by antiapoptotic activity [bib_ref] Cardioprotective effects of selective inhibition of the two complement activation pathways in..., Murohara [/bib_ref] [bib_ref] Blocking of classical complement pathway inhibits endothelial adhesion molecule expression and preserves..., Buerke [/bib_ref] [bib_ref] Anti-ischemia/reperfusion of C1 inhibitor in myocardial cell injury via regulation of local..., Fu [/bib_ref] [bib_ref] Anti-apoptotic role for C1 inhibitor in ischemia/reperfusion-induced myocardial cell injury, Fu [/bib_ref]. However, it remains to be evaluated whether similar events de facto occur in cardiac dysfunction during systemic inflammation. ## Hepatic failure The liver represents the "major production facility" for most complement proteins found in the blood compartment except C1q, factor D, and properdin [bib_ref] Complement and complement deficiencies, Whaley [/bib_ref]. Because of its integral role in metabolism and host defense, the liver plays a key role in the initiation of MODS [bib_ref] Kupffer cells are responsible for producing inflammatory cytokines and hepatocellular dysfunction during..., Koo [/bib_ref] [bib_ref] The liver as an immune organ, Sheth [/bib_ref]. Enhanced interaction of leukocytes with hepatic endothelial cells and hepatic microperfusion disorders are fundamental contributors to liver failure during sepsis [bib_ref] C1-inhibitor reduces hepatic leukocyteendothelial interaction and the expression of VCAM-1 in LPS-induced..., Croner [/bib_ref]. Like in other organs, complement activation products are generated among other inflammatory mediators during systemic inflammation, which initiate a cascade of intracellular events in target cells leading to upregulation of adhesion molecules (ICAM-1, VCAM-1) on hepatic epithelial cells, increase of vascular permeability, and priming and influx of leukocytes [bib_ref] C1-inhibitor reduces hepatic leukocyteendothelial interaction and the expression of VCAM-1 in LPS-induced..., Croner [/bib_ref] [bib_ref] Cellular adhesion molecules: regulation and functional significance in the pathogenesis of liver..., Jaeschke [/bib_ref]. Treatment with C1 inhibitor reduced VCAM expression and hepatic leukocyte adhesion in experimental acute hepatic failure, even after delayed injection [bib_ref] C1-inhibitor reduces hepatic leukocyteendothelial interaction and the expression of VCAM-1 in LPS-induced..., Croner [/bib_ref]. Besides this mechanism, PMN mediate parenchymal damage after accumulation in sinusoids, which does not depend on cellular adhesion molecules [bib_ref] Sequestration of neutrophils in the hepatic vasculature during endotoxemia is independent of..., Jaeschke [/bib_ref]. The liver is not only the main source of complement proteins but is also constantly exposed to complement-activating pathogens via the portal venous system [bib_ref] Intrinsic resistance of hepatocytes to complement-mediated injury, Koch [/bib_ref] [bib_ref] Endotoxin and bacteria in portal blood, Jacob [/bib_ref]. Immune complexes, anaphylatoxins, and activated complement components are cleared from circulation by the reticuloendothelial system lining the sinusoids without being detriment to hepatic function [bib_ref] Complement and complement deficiencies, Whaley [/bib_ref]. However, the efficiency of the reticuloendothelial system does not suffice to protect the liver. Therefore, hepatocytes are endowed with a unique mechanism to protect themselves from complement-induced cytotoxicity [bib_ref] Intrinsic resistance of hepatocytes to complement-mediated injury, Koch [/bib_ref]. It is intriguing that this protection is not dependent on the complement regulatory proteins on the cell surface [bib_ref] Intrinsic resistance of hepatocytes to complement-mediated injury, Koch [/bib_ref]. Instead, the inurement of hepatocytes to complement and its activated products requires the integrity of the PI3K/Akt pathway [bib_ref] Intrinsic resistance of hepatocytes to complement-mediated injury, Koch [/bib_ref]. In turn, the PI3K/Akt pathway supposedly controls C5a-mediated effects in PMN and monocytes [bib_ref] The phosphatidylinositol 3-kinase signaling pathway exerts protective effects during sepsis by controlling..., Wrann [/bib_ref]. In experimental sepsis, anti-C5a treatment circumvented the development of MOF and attenuated markers of acute hepatic failure (e.g., bilirubin, ALT, AST, LDH) [fig_ref] Figure 1: Summarizing illustration on the effects of excessive complement activation on various organ... [/fig_ref] [bib_ref] Role of C5a in multiorgan failure during sepsis, Huber-Lang [/bib_ref]. Thus, it is tempting to speculate that under conditions, in which C5a is systemically generated, impairment of the PI3K/Akt pathway may lead to increased susceptibility for complement-mediated cytotoxicity of hepatocytes and subsequent organ failure. On the other hand, a potential role for C5a in tissue repair has been suggested [bib_ref] Role of C5a in inflammatory responses, Guo [/bib_ref]. ## Renal failure Acute renal failure (ARF) is hallmarked by abrupt decline in glomerular filtration and acute tubular necrosis in association with the appearance of multiple inflammatory mediators [bib_ref] Complement is activated in kidney by endotoxin but does not cause the..., Cunningham [/bib_ref] [bib_ref] Role of soluble mediators in sepsis and renal failure, Camussi [/bib_ref] [bib_ref] Complement activation in patients with renal failure as detected through the quantitation..., Oppermann [/bib_ref]. In sepsis, ARF occurs already at modest levels of hypotension suggesting that other mechanisms than ischemia are involved [bib_ref] Complement is activated in kidney by endotoxin but does not cause the..., Cunningham [/bib_ref]. Like in parenchymal cells of lung and brain, complement proteins can be locally produced by renal cells, such as proximal tubular cells, in vitro and in vivo [bib_ref] Cellular specificity of murine renal C3 expression in two models of inflammation, Ault [/bib_ref] [bib_ref] Interleukin 2 mediates stimulation of complement C3 biosynthesis in human proximal tubular..., Brooimans [/bib_ref]. In the case of C3, there is evidence that its renal production even contributes to the circulating C3 pool [bib_ref] Contribution of renal secreted complement C3 to the circulating pool in humans, Tang [/bib_ref]. Proximal tubular cells are capable of activating the alternative pathway, terminating in the binding of MAC to the cell surface [bib_ref] Alternative pathway activation of complement by cultured human proximal tubular epithelial cells, Biancone [/bib_ref]. In this context, it is of particular interest that the luminal brush border lacks complement regulatory proteins on the cell surface [bib_ref] Localization of the complement regulatory proteins in the normal human kidney, Ichida [/bib_ref]. Under certain circumstances, paucity of protection against complementmediated cell lysis predisposes to tubular damage due to the luminal deposition of filtered complement components [bib_ref] Acute tubular necrosis is characterized by activation of the alternative pathway of..., Thurman [/bib_ref]. The deposition of C3 and C4 is well established in glomerular disease, but only C3 deposition, and no evidence for C4 deposition, along tubules could be found in acute tubular necrosis after renal ischemia/reperfusion injury, indicating that the alternative pathway is the predominant complement activation pathway for the development of acute tubular necrosis [bib_ref] Acute tubular necrosis is characterized by activation of the alternative pathway of..., Thurman [/bib_ref]. However, suppression of C3 activation failed to affect the degree of ARF in a murine model of systemic inflammation, although C3 synthesis was upregulated, resulting in basolateral tubular C3 deposition [bib_ref] Complement is activated in kidney by endotoxin but does not cause the..., Cunningham [/bib_ref]. In disagreement with these authors' conclusion, this does not necessarily mean that complement is not responsible for ARF in the setting of systemic inflammation since it is now known that the downstream complement cascade can be activated despite the absence of C3 [bib_ref] Generation of C5a in the absence of C3: a new complement activation..., Huber-Lang [/bib_ref]. In contrast, the occurrence of ARF could be clearly linked to the generation of C5a during experimental sepsis, and parameters of ARF (creatinine, urine output, glomerular filtration rate, proteinuria) as well as morphological changes of podocytes were greatly attenuated by anti-C5a treatment [bib_ref] Role of C5a in multiorgan failure during sepsis, Huber-Lang [/bib_ref]. Beyond their local inflammatory and chemotactic features, C3a and C5a have vascular effects that contribute to changes in renal hemodynamics in ARF [fig_ref] Figure 1: Summarizing illustration on the effects of excessive complement activation on various organ... [/fig_ref] [bib_ref] Endotoxin-induced shock in the rat. A role for C5a, Smedegard [/bib_ref]. Taken together, the complement system represents a key effector of ARF by a variety of mechanisms, which affect renal perfusion and glomerular filtration as well as tubular function. ## Dysregulation of the coagulation system The coagulation system and the complement system are both proteolytic cascades composed of serine proteases that share structural characteristics. As descendants of a common ancestor, both systems can be basically activated by similar stimuli [bib_ref] Evolution of enzyme cascades from embryonic development to blood coagulation, Krem [/bib_ref] [bib_ref] The impact of the inflammatory response on coagulation, Esmon [/bib_ref]. Trauma and tissue injury often cause damage of the vasculature and subsequent bleeding, which is also associated with the risk of infection by intruding microorganisms [bib_ref] Pathophysiology of polytrauma, Keel [/bib_ref]. Activation of both cascades is intended to occur locally under thorough regulations, but under certain circumstances, loss of control can lead to systemic activation with harmful consequences for the host [bib_ref] Complement and coagulation: strangers or partners in crime?, Markiewski [/bib_ref]. Disseminated intravascular coagulation (DIC) represents a frequent complication after trauma, systemic inflammation, and sepsis [bib_ref] Combined activation of coagulation and inflammation has an important role in multiple..., Gando [/bib_ref] [bib_ref] New treatment strategies for disseminated intravascular coagulation based on current understanding of..., Levi [/bib_ref]. After the initial phase of hypercoagulability with intra-and extravascular fibrin clots, consumption of coagulation factors and dysfunction of thrombocytes can lead to hemorrhagic diasthesis and diffuse bleeding [bib_ref] Coagulation abnormalities in acute lung injury and sepsis, Abraham [/bib_ref] [bib_ref] Combined activation of coagulation and inflammation has an important role in multiple..., Gando [/bib_ref] [bib_ref] New treatment strategies for disseminated intravascular coagulation based on current understanding of..., Levi [/bib_ref]. Intravascular fibrin clots are finally responsible for impaired microcirculation and hypoxic cellular damage [bib_ref] Coagulation abnormalities in acute lung injury and sepsis, Abraham [/bib_ref]. Trauma, thermal injury, and infection predispose to thrombosis and the development of DIC and trigger the inflammatory response including complement activation, which, in turn, can trigger coagulation and vice versa [bib_ref] The impact of the inflammatory response on coagulation, Esmon [/bib_ref] [bib_ref] New treatment strategies for disseminated intravascular coagulation based on current understanding of..., Levi [/bib_ref]. As mentioned above, thrombin is capable of cleaving C5, resulting in the generation of C5a. This concept of a direct crosstalk between central components of the complement and coagulation cascades is corroborated by the findings of elevated thrombin-antithrombin (TAT) complexes in the clinical and experimental setting of multiple injury [bib_ref] Molecular intercommunication between the complement and coagulation systems, Amara [/bib_ref]. Beside the C5-convertase activity of thrombin, various factors of the coagulation and fibrinolysis system, including FXa, FXIIa, plasmin, and kallikrein, can cleave complement components or their fragments [bib_ref] Generation of C5a in the absence of C3: a new complement activation..., Huber-Lang [/bib_ref] [bib_ref] Activation of the classical pathway of complement by Hageman factor fragment, Ghebrehiwet [/bib_ref] [bib_ref] NH2-terminal structure and cleavage of guinea pig pro-C3, the precursor of the..., Goldberger [/bib_ref] [bib_ref] C3d-K, a kallikrein cleavage fragment of iC3b is a potent inhibitor of..., Thoman [/bib_ref]. On the other hand, the inflammatory response and the complement system in particular amplify coagulation by modification of phospholipid membranes required for the initiation of the tissue factor (TF) pathway, activation of platelets, and upregulation of TF expression [bib_ref] The impact of the inflammatory response on coagulation, Esmon [/bib_ref]. Specifically, activation of C5 can increase TF expression on leukocytes and blockade of C5a-ameliorated DIC in a rodent model of sepsis [bib_ref] Anti-C5a ameliorates coagulation/fibrinolytic protein changes in a rat model of sepsis, Laudes [/bib_ref] [bib_ref] C5 chemotactic fragment induces leukocyte production of tissue factor activity. A link..., Muhlfelder [/bib_ref]. The procoagulant activities of complement are aggravated by inhibition of anticoagulant mechanisms, such as complex formation of C4b-binding protein with protein S (PS), which results in a loss of PS cofactor activity for activated protein C (APC) [bib_ref] Coagulation, inflammation, and apoptosis: different roles for protein S and the protein..., Rezende [/bib_ref]. In turn, the protein C anticoagulant pathway does not only function as a regulator of the coagulation cascade by degradation of FVa and FVIIIa, but also dampens the inflammatory response [bib_ref] The impact of the inflammatory response on coagulation, Esmon [/bib_ref] [bib_ref] Efficacy and safety of recombinant human activated protein C for severe sepsis, Bernard [/bib_ref]. Traditionally, complement and coagulation were described as separate cascades, only linked by the ability of FXIIa to activate the classical complement pathway [bib_ref] Activation of the classical pathway of complement by Hageman factor fragment, Ghebrehiwet [/bib_ref]. However, it becomes now more and more evident that the convergence between both systems extends beyond the biochemical nature of serine proteases, and multiple mutual interconnections form a highly complex network [fig_ref] Figure 1: Summarizing illustration on the effects of excessive complement activation on various organ... [/fig_ref] [bib_ref] Complement and coagulation: strangers or partners in crime?, Markiewski [/bib_ref] [bib_ref] Interaction between the coagulation and complement system, Amara [/bib_ref] [bib_ref] Molecular intercommunication between the complement and coagulation systems, Amara [/bib_ref]. Understanding the interplay is important to breach the vicious circle of systemic inflammation in order to be able prevent life-threatening complications. # Conclusions Based upon the current understanding, the general role of complement in the pathogenesis of MOF can be conceptualized as follows: After trauma, burn, or severe tissue injury, systemic intravascular activation of the complement system with apparent loss over the control mechanisms occurs. Complement activation products trigger a cascade of cellular events in endothelial cells resulting in upregulation of adhesion molecules, release of proinflammatory mediators, and increased vascular permeability. Leukocytes are attracted by complement anaphylatoxins to transmigrate into parenchyma of various organs after adhesion to endothelial cells and extravasation. Activated leukocytes release inflammatory mediators, enzymes, and free radicals that harm parenchymal cells. Local production and activation of complement proteins in combination with loss of protection against complement-mediated lysis aggravate the degree of tissue injury. Interaction with the coagulation cascade causes disseminated intravascular coagulation and compromised microcirculation, which then augments organ dysfunction by ischemia. All events of this vicious circle finally merge into apoptosis and necrosis of parenchymal cells with the development of multiple organ dysfunction syndrome. The complement anaphylatoxins C5a and C3a not only trigger the inflammatory response but also directly alter cellular functions of parenchymal cells as well as leukocytes by interaction with their specific receptors, which are abundantly expressed on numerous cell types. However, the organ-specific mechanisms and intracellular events that follow receptor binding, such as mitogen-activated protein kinase (MAPK) pathways, remain to be evaluated in future studies. As outlined above in the description of cardiac dysfunction, organ failure might reflect a cellular resting state, also described as hibernation, as a response to a proinflammatory environment with uncoupling of the respiratory chain and mitochondrial dysfunction. However, it is not clear yet if and to which extent complement activation contributes to the pathophysiology of hibernation in human cells. Since complement activation occurs as a rapid event after the initial insult, it appears auspicious to use intervention in the complement system as a therapeutic approach in order to prevent the development of MOF. Strategies to inhibit complement include (i) the application of endogenous complement inhibitors (C1 inhibitor, soluble complement receptor-1) [bib_ref] C1-inhibitor in patients with severe sepsis and septic shock: beneficial effect on..., Caliezi [/bib_ref] , (ii) administration of antibodies or antagonists which block key proteins (C3, C5) of the complement cascade or neutralize complement-derived anaphylatoxins (C3a, C5a) [bib_ref] Protective effects of C5a blockade in sepsis, Czermak [/bib_ref] [bib_ref] Complement inhibition decreases the procoagulant response and confers organ protection in a..., Silasi-Mansat [/bib_ref] , and (iii) interference of C5a, C3a interaction with their receptors by receptor-specific antagonists [bib_ref] Functional roles for C5a receptors in sepsis, Rittirsch [/bib_ref]. In addition, upregulation or incorporation of membranebound complement-regulatory proteins could protect organs from complement-mediated cytotoxicity. Protection against complement-mediated inflammatory tissue damage could be achieved in various experimental settings. However, total blockade of the complement cascade might impair the capability to clear invaded pathogens and increase the risk of infection. Therefore, targeting the complement system in inflammation should rather aim to balance or control its activation with suppression of the harmful effects, but without detriment of the protective and reparative complement functions. [fig] Figure 1: Summarizing illustration on the effects of excessive complement activation on various organ systems and the development of organ failure. For details see text. MBL: mannose-binding lectin, CNS: central nervous system, BBB: blood brain barrier, PMN: polymorphonuclear neutrophils, ARDS: acute respiratory distress syndrome, ATIII: antithrombin III, RES: reticuloendothelial system, GFR: glomerular filtration rate, ATN: acute tubular necrosis, FX: coagulation factor X, FII: coagulation factor II, TF: tissue factor, DIC: disseminated intravascular coagulation. [/fig]

Grade IV blunt splenic injury – the role of proximal angioembolization. A case report and review of literature The authors present a case of grade IV traumatic spleen rupture (AAST-OIS) and an Injury Severity Score of 21 and a Revised Trauma Score RTS=7.841, which was managed without surgery, but with proximal splenic angioembolization (SAE), with a positive outcome. Indications, types and side-effects of SAE are also discussed with regard to blunt spleen trauma and the benefits of SAE as non-operative treatment approach. It is the first case of a grade IV splenic laceration non-operatively managed to be published in Romania.Keywords: severe splenic laceration, non-operative management, angio-embolizationThe treatment of blunt splenic trauma has significantly changed over the last decades, when the non-operative management approach was first employed. SAE, as part of the NOM protocol, has enabled surgeons to treat many patients with severe blunt splenic injuries (grade III, IV) who are hemodynamically stable or easy to stabilize in a conservative manner. Thanks to SAE, there has been an increase in the number of patients treated non-operatively and a decrease of therapy-associated failures (due to undiagnosed splenic vascular injuries that progressed).Non-operative management of blunt splenic trauma is the safest method to preserve the organ itself and to prevent the unwanted consequences which occur after spleen removal. ) after a fall from height (8 meters) with multiple trauma, haemoperitoneum after splenic rupture, blunt bladder injury with microscopic hematuria, mild cerebral contusion (retrograde amnesia), facial abrasions, blunt chest trauma and blunt right knee injury.Past medical history included hepatitis C virus infection.On admission: GCS=15, retrograde amnesia, hemodynamically stable (BP=122/70 mmHg; HR=75/min, sinus rhythm; RR=14/min), abdomen soft, no rebound or guarding, tender in the left upper quadrant and epigastrium. The abdominal ultrasound showed perisplenic fluid (3/15 mm) and a hypoechoic splenic area of 2/12 mm.Contrast CT showed extensive splenic laceration exceeding 60% of the spleen, sparing the upper pole, with capsule disruption but sparing the splenic pedicle, perihepatic and perisplenic haemoperitoneum(Fig. 1, 2); blood in the pelvis(Fig. 3). Fig. 1 CT scan showing grade IV splenic laceration; perisplenic, perihepatic hemoperitoneum. The presence of a grade IV splenic injury (AAST-OIS) with a moderate haemoperitoneum imposed the performance of splenic angiography that revealed a heterogeneous contrast uptake within the splenic parenchyma with fine areas of contrast extravasation [fig_ref] Figure 4: Splenic angiography -heterogeneous contrast uptake within the splenic parenchyma with fine areas... [/fig_ref]. A proximal SAE was performed by using a fibrin sealant TachoSil ®; a final angiographic check did not highlight any other areas of contrast extravasation. [fig_ref] Figure 5: Splenic angioembolization -postprocedural image [/fig_ref] The post-procedure progress was very good. A repeat CT exam on day 9 showed a normal postembolization aspect. A peripheral blood smear taken on day 10 post-embolization did not show any Howell-Jolly antibodies. # Discussion According to Lucas [bib_ref] Choice of Management, Lucas [/bib_ref] , the initiator of nonoperative management of splenic trauma was Wanborough, in 1940 (Sick Children's Hospital Toronto). In 1968, Upadhyaya said that ˝ ... very often, in children with splenic trauma, a significant blood loss is not apparent. It is interesting that the majority of children in this series had no splenic bleeding at the time of laparotomy ˝ [bib_ref] Tribute to a Triad: History of Splenic Anatomy, Physiology, and Surgery -Part..., Mcclusky [/bib_ref]. This is explained by various mechanisms: hypotension, clots, regional blocking by the greater omentum, containment and local pressure effect produced by the newly developed perisplenic hematoma and the presence of an intact splenic capsule. In 1971, Douglas and Simpson from the Toronto Hospital for Sick Children, described 32 children with clinical signs of splenic injury, who were non-operatively treated; 25 of them did not require surgery. This study proved that an injured spleen can heal spontaneously and in most cases, the recovery being uneventful [bib_ref] President´s Address. Caring for the Major Trauma Victim: the Role of Radiology, Mccort [/bib_ref]. In traumatic spleen injuries in adults, the surgeons were initially reluctant in selecting the nonoperative treatment for the following reasons: the postsplenectomy sepsis was less common and less dangerous than in children; age-related architectural and vascular changes within the spleen are less likely to produce a spontaneous hemostasis; the risk of omitting associated lesions; the possibility of a DRS (delayed rupture of the spleen) and the occurrence of posttraumatic pseudocyst or splenosis [bib_ref] Non-operative management of blunt splenic injury -works well in about a quarter..., Gibney [/bib_ref]. Patients with traumatic splenic injuries can be treated surgically or non-operatively, according to the patient's, surgeon's or hospital's characteristics. Britt [bib_ref] ˝Alternative ˝ surgery in trauma management, Britt [/bib_ref] introduced the term "alternative surgery" in order to define the non-operative treatment or the selective approach of trauma patients. NOM is the updated concept of SOS-"Save Our spleens " -(originally applied to children) and one which, some surgeons have had the courage to apply to adults. The situation has changed after Sclafani performed a successful proximal splenic angioembolization in adult patients with spleen trauma [bib_ref] The role of angiographic hemostasis in salvage of the injured spleen, Sclafani [/bib_ref] [bib_ref] Blunt splenic injuries: nonsurgical treatment with CT, arteriography, and transcatheter arterial embolization..., Sclafani [/bib_ref] [bib_ref] Nonoperative salvage of computed tomographydiagnosed splenic injuries: utilization of angiography for triage..., Sclafani [/bib_ref]. The method was subsequently used in children but with a much lower frequency and indications yet unclear. Currently, it is considered that angiography (both diagnostic and therapeutic) is part of splenic NOM algorhythm for spleen trauma [bib_ref] The evolving role of interventional radiology in trauma care, Pryor [/bib_ref] [bib_ref] Splenic artery embolization: Have we gone too far, Smith [/bib_ref] [bib_ref] Angioembolization reduces operative intervention for blunt splenic injury, Wei [/bib_ref]. The method is indicated in hemodynamically stable patients or easy to stabilize. Hypotension upon admission is a fiercely debated issue particularly because it may be caused by various factors. Hagiwara [bib_ref] Nonsurgical management of patients with blunt splenic injury: efficacy of transcatheter arterial..., Hagiwara [/bib_ref] and Bee [bib_ref] Failures of splenic nonoperative management: is the glass half empty or half..., Bee [/bib_ref] showed that hypotension (as a single parameter) is not a prognostic factor for the failure of NOM. Similarly, Chen [bib_ref] Spleen artery embolization increases the success of nonoperative management following blunt splenic..., Chen [/bib_ref] stressed that hypotension, if correctly interpreted and treated, is not a contraindication for SAE. Indications for splenic angiography are the following [bib_ref] Nonoperative management of blunt splenic injury: a 5-year experience, Haan [/bib_ref] [bib_ref] Blunt splenic injuries: high nonoperative management rate can be achieved with selective..., Dent [/bib_ref] [bib_ref] Early selective angioembolization improves success of nonoperative management of blunt splenic injury, Wu [/bib_ref] [bib_ref] The effects of splenic artery embolization on nonoperative management of blunt splenic..., Sabe [/bib_ref] : € splenic injury grades 3, 4, 5; € vascular lesions seen on the initial CT, regardless of severity; € active bleeding obvious on CT or "contrast blush" in hemodynamically stable patients; € an unexplained Hct decline in the absence of other injuries; € severe haemoperitoneum. SAEs increased the NOM's success by stopping the bleeding and preventing the delayed rupture of the spleen [bib_ref] Impact of splenic artery embolization on the success rate of nonoperative management..., Van Der Vlies [/bib_ref]. Currently, various materials have been used for splenic embolization, such as metallic coils, fragments of hemostatic agents ("Gelfoam pledgets", TachoSil ®) with a diameter of more than 1,000 μm, which injected through the catheter occlude the vessel achieving hemostasis (special MRI-compatible coils were created which would allow further MRI examinations) or microspheres. The main advantage of these hemostatic agents is that after a few weeks, they are reabsorbed through the action of macrophages, thus achieving repermeabilization of the blood vessel [bib_ref] The role of arterial embolization in blunt splenic injury (Il ruolo dell'embolizzatione..., Franco [/bib_ref]. However, due to this exact characteristic, some authors [bib_ref] The role of arterial embolization in blunt splenic injury (Il ruolo dell'embolizzatione..., Franco [/bib_ref] [bib_ref] Use of splenic artery embolization as an adjunct to nonsurgical management of..., Liu [/bib_ref] [bib_ref] Experience with splenic main coil embolization and significance of new or persistent..., Haan [/bib_ref] actually contraindicate these hemostatic agents (increased rate of re-bleeding). Similarly, Smith [bib_ref] Splenic artery embolization: Have we gone too far, Smith [/bib_ref] reported excellent results with the use of metal coils. Haan [bib_ref] Implications of the "contrast blush" finding on computed tomographic scan of the..., Omert [/bib_ref] reported an increased frequency of splenic infarction after using Gelfoam. Finding traumatic intra-splenic vascular lesions is still an important topic of debate. Omert et al [bib_ref] Implications of the "contrast blush" finding on computed tomographic scan of the..., Omert [/bib_ref] considered the "contrast blush" a common finding in severe splenic injuries, yet it does not represent a surgical indication per se. Contrast blush is defined as a contrast focusing within the parenchyma or extra-capsular found on early or late CT films. Burlew et al [bib_ref] Blunt trauma induced splenic blushes are not created equal, Burlew [/bib_ref] found that intrasplenic contrast extravasation is not an absolute indication for SAE / surgery. The study was based on the finding that not all trauma patients transferred from Denver Health Medical Center for spleen angioembolization had contrast blush on repeat CT examination. Therefore, not all contrastblushes are equivalent, some resolving spontaneously through intra-parenchymal tamponade effect. This result should, however, be regarded with some reservation due to the retrospective character of the study, the small number of cases and the lack of specification regarding the CT exam parameters (the amount of contrast administered, infusion rates, section thickness, late images). The major problem with this algorhythm is patient selection criteria (repeat CT exam?, Doppler Ultrasound?, Ultrasound with contrast?). Omert [bib_ref] Implications of the "contrast blush" finding on computed tomographic scan of the..., Omert [/bib_ref] states that the patient's hemodynamic parameters have a higher predictability than the isolated presence of contrast-blush to establish therapeutic indications; the mere presence of contrast-blush is not an absolute indication for SAEs. A similar result has been reported in pediatric traumatic spleen pathology [bib_ref] Pediatric splenic injuries with a contrast blush: successful nonoperative management without angiography..., Cloutier [/bib_ref]. The authors' recommendations in these cases are: repeat, careful, clinical and laboratory observations, dynamic FAST (especially in cases with initial negative FAST ultrasound results) and, in some cases, abdominal CT exam. Recently Michailidou and Velmahos [bib_ref] Blush" on trauma computed tomography: Not as bad as we think!, Michailidou [/bib_ref] found in a retrospective study that not all contrast extravasationes (as shown at MDCT-64 multidetector scanner) require interventional radiology / surgery. These high-speed scanners can identify very small contrast extravasations and with current unclear significance. The authors evaluated IV contrast according to location (organ, pelvis and other locations), to size (small <1.5 cm large ≥ 1.5 cm), free peritoneal fluid versus intraparenchymal blush and according to number (single, multiple); a late blush was considered at 3 hours after admission. Finally, 43.5% of the cases did not require any therapeutic intervention. The statistical analysis revealed three independent factors that were highly predictive for therapeutic intervention: SBP ≤ 100 mmHg upon admission, large blush and abdominal AIS ≥ 3; simultaneous presence of all 3 was associated with therapeutic intervention in 100% of the cases whilst their absence was associated with an intervention in 31% of cases. A very interesting finding was that 73% of the splenic injuries with contrast blush required therapeutic interventions and of these, 50% had small blushes (<1.5 cm) and 82% had large blushes (≥ 1.5 cm). Bhullar [bib_ref] At first blush: Absence of computed tomography contrast extravasation in grade IV..., Bhullar [/bib_ref] noticed that out of 1056 patients with blunt spleen trauma, 95 (17%) had contrast blush on CT examination and in 97.7% blood extravasation was confirmed on angiography. 9.5% of the patients treated non-operatively had severe lesions (grades IV-V) without contrast blush on CT out of which, 20 (85%) had angiography-proven active bleeding. For these patients, SAE has been successful, however, in 26% of those cases with severe injuries and without contrast blush and without splenic angiography, NOM was a failure. The author concluded that the SAE is required in all splenic contusions with contrast blush, regardless of the lesion severity. Also, in severe splenic injuries (grades IV-V) managed with NOM, SAE is required, even without contrast blush on CT scan. The final conclusion belongs to Scale (Michailidou-discussion 26): as not all organs are identical, current MDCT shows vascular lesions that would not have been observed in the past, but whose therapeutic value were questionable / minor. Thus, we can only agree with Marmery [bib_ref] Optimization of selection for nonoperative management of blunt splenic injury: comparison of..., Marmery [/bib_ref] , who proposed a new CT classification of traumatic spleen injuries, which would include active bleeding, arterio-venous intrasplenic fistula, pseudoaneurysm and vascular lesions as essential elements for the correct assessment of injury extent. Willman (cit. 26) reports that "when IV contrast extravasation is found, surgery /immediate therapeutic angiography is needed. Fangreports that in the presence of IV contrast extravasation "non-operative treatment should be stopped and emergency angiography / surgery should be performed without delay." Have these findings become history? Michailidou [bib_ref] Blush" on trauma computed tomography: Not as bad as we think!, Michailidou [/bib_ref] showed that most of these findings are based on large blushes (≥ 1.5 cm), which were, in fact, the only ones detected with CT scanners at that time and which would, obviously, suggest an active bleeding. These findings are no longer accurate in the era of high-speed MDCT, which can highlight minimal bleeding with little/ no consequences. Thus, the authors' conclusion is similar to Omert's [bib_ref] Implications of the "contrast blush" finding on computed tomographic scan of the..., Omert [/bib_ref] : "the presence of contrast blush is not an absolute indication for surgery / angiography". Clinically, the SAE should be performed as soon as the contrast extravasation is found (abdominal CT examination) and before the onset of hemodynamic instability [bib_ref] Early selective angioembolization improves success of nonoperative management of blunt splenic injury, Wu [/bib_ref]. According to Schurr's [bib_ref] Management of blunt splenic trauma: computed tomographic contrast predicts failure of nonoperative..., Schurr [/bib_ref] and Bhullar [bib_ref] At first blush: Absence of computed tomography contrast extravasation in grade IV..., Bhullar [/bib_ref] the presence of contrast blush increases the risk of NOM failure by 22 to 24 fold. SAE may be carried out: - in an emergency ("acute")-in hemodynamically stable patients with active bleeding or severe splenic injuries (III-V); - delayed ("subacute")-indications are represented by declining of Hb values during NOM or finding a pseudoaneurysm (> 1.5 cm) or the occurrence of a contrast blush on repeat CT examination. Splenic angio-embolization (SAEs) can be carried out: - distally (supra-selective)-achieves containment of damaged vessel, retains a normal blood flow to an important splenic area but is a time-consuming procedure and requires high technical skills [bib_ref] Is anything new in adult blunt splenic trauma, Harbrecht [/bib_ref]. - Proximally (the trunk of splenic artery, distal to the dorsal pancreatic artery) is performed with metal coils or absorbable hemostatic materials (Gelfoam-Pharmacia, Kalamazoo,MI; Tacho-comb) and achieves hemostasis by decreasing arterial blood flow and intra-splenic pressure, which promotes clot formation and wound healing. The spleen viability is ensured by collateral circulation (branches of the left gastric artery, gastroepiploic arteries, omental, pancreatic, short gastric arteries) as shown by experimental animal studies (12, Anderson cit. 32). In human studies, it was found that there was a decline of intrasplenic pressure by 47-58% (Bessoud cit. . Sclafani [bib_ref] Nonoperative salvage of computed tomographydiagnosed splenic injuries: utilization of angiography for triage..., Sclafani [/bib_ref] believes that this procedure is compatible with the maintenance of splenic immune function and even if surgery is necessary, splenorrhaphy is facilitated. Proximal SAE is faster, easier to perform and is associated with a lower NOTrelated failure rate and a reduced incidence of post-procedural complications (abscess, splenic infarction) [bib_ref] Splenic artery embolization: Have we gone too far, Smith [/bib_ref] [bib_ref] Impact of splenic artery embolization on the success rate of nonoperative management..., Van Der Vlies [/bib_ref] [bib_ref] Experience with splenic main coil embolization and significance of new or persistent..., Haan [/bib_ref] [bib_ref] Implications of the "contrast blush" finding on computed tomographic scan of the..., Omert [/bib_ref] [bib_ref] CT findings after embolization for blunt splenic trauma, Killeen [/bib_ref]. According to Van der Vlies [bib_ref] Impact of splenic artery embolization on the success rate of nonoperative management..., Van Der Vlies [/bib_ref] , the only drawback of proximal SAE is that, in the event of a possible re-bleeding, supraselective embolization is difficult / impossible to perform. The lack of highlighting of left gastric artery or its tributaries on angiography may be a cause of proximal post -SAE splenic infarction [bib_ref] Proximal splenic artery embolization in blunt splenic trauma, Zmora [/bib_ref]. This can be explained by compromised collateral circulation secondary to agerelated splenic pathology. It should be noted that the presence of a patent left gastric artery may be of particular importance to maintaini collateral circulation after proximal splenic SAE [bib_ref] Proximal splenic artery embolization in blunt splenic trauma, Zmora [/bib_ref]. For this reason, it is indicated that a celiac trunk arteriography is obtained with special focus on the left gastric artery [bib_ref] Splenic artery embolization in blunt trauma. How I do it, Imbrogno [/bib_ref]. Splenic artery occlusion was originally used to control hypersplenism associated with liver cirrhosis and portal hypertension syndrome. Hypersplenism recurrence suggests the development of collateral circulation, which in Harbrecht's opinion [bib_ref] Is anything new in adult blunt splenic trauma, Harbrecht [/bib_ref] , would allow for the traumatized spleen to keep its functions. - Combined Angiographic examination is performed after the CT scan revealed intra-splenic vascular lesions in order to confirm and potentially embolize them at the same time. Embolization is performed only on the basis of angiographic lesion diagnosis [bib_ref] Nonoperative management of blunt splenic injury: a 5-year experience, Haan [/bib_ref] [bib_ref] Splenic artery embolization in blunt trauma. How I do it, Imbrogno [/bib_ref]. After embolization, patients were admitted to ICU, where their vitals, blood count (dynamic values of hemoglobin) and clinical status (repeat abdominal examinations) were closely monitored. There are views who argue that proximal SAE causes fewer complications and increases the NOM's success rate (by decreasing blood flow and intrasplenic pressure -47-58% -facilitates clot formation and the collateral circulation ensures viability of the spleen). SAE may cause distal necrosis / infection, in keeping with the avascular area. A tortuous splenic artery makes distal angioembolization difficult / impossible, thus being an indication for proximal SAE only. Repeated embolization ("second-look" angiography) is indicated in recurrent bleeding and after an initial negative angiography (10%) [bib_ref] Admission angiography for blunt splenic injury:advantages and pitfalls, Haan [/bib_ref]. Haan is partial to distal embolization for mild and combined splenic injuries (with no significant statistical differences). According to the same author [bib_ref] Admission angiography for blunt splenic injury:advantages and pitfalls, Haan [/bib_ref] "delayed vascular emergencies" (term introduced by the Memphis group) are actually diagnostic delays which may be shown by angiography in severe splenic lesions (grade 3,4,5). The Memphis Group (Davis, Fabian, Croce) showed that there may be vascular lesions, initially not identified on CT or angiographic exams due to arterial spasm at the time of the examination, and subsequently become symptomatic. Repeated spiral CT identified 80% of the initially missed vascular lesions (used as a screening test for angiography). The only statistically significant marker of NOM failure is the arterio-venous fistula for which the proximal embolization is not sufficient, requiring a more direct approach-the distal embolization [bib_ref] Role of embolization in the management of splenic trauma, Lui [/bib_ref]. The findings of Haan [bib_ref] Western Trauma Association Multi-Institutional Trials Committee. Splenic embolisation revisited: a multicenter review, Haan [/bib_ref] are: € proximal embolization (decreases splenic perfusion pressure) is a therapeutic modality much more useful than distal embolization, except for the AV fistula; € the immunological consequences of proximal embolization are unclear and require further study; € the use of SAE decreases by 20% the failure rate of NOM in grades 4 and 5 splenic injuries; € the SAE would be superior to surgery in the treatment of blunt splenic injuries in multiple trauma patients with head injuries. € SAE is a useful and effective method as part of the NOM protocol but employed in just 7% of cases. Haan [bib_ref] Experience with splenic main coil embolization and significance of new or persistent..., Haan [/bib_ref] abandoned selective SAE in favor of proximal SAE, performed with metal coils, however finding a new category of patients: with persistent postprocedural pseudoaneurysm (PSA) or new postprocedural PSA. In these patients, the success rate of NOM was 91% and the spleen preservation was achieved in 94% of cases. The indications of SAE are [bib_ref] The role of arterial embolization in blunt splenic injury (Il ruolo dell'embolizzatione..., Franco [/bib_ref] [bib_ref] Experience with splenic main coil embolization and significance of new or persistent..., Haan [/bib_ref] [bib_ref] Role of embolization in the management of splenic trauma, Lui [/bib_ref] [bib_ref] Nonoperative management of splenic injuries: improved results with angioembolization, Gaarder [/bib_ref] : - combined SAE: multiple vascular lesions (severe injury degrees), intraperitoneal extravasation. After SAE, repeat CT (postprocedure) focuses on finding: persistent vascular injury, pseudoaneurysm formation, infracted area size, evidence of local infection (splenic abscess). [formula] - [/formula] Hagiwara [bib_ref] Nonsurgical management of patients with blunt splenic injury: efficacy of transcatheter arterial..., Hagiwara [/bib_ref] recommends a repeated CT scan (in patients with SAE) between days 10 and 15 post-SAE in order to assess perfusion and splenic blood flow through collateral circulation. The splenic reticuloendothelial function was investigated by scintigraphy with colloid sulfur Tc-99m having been performed between days 10 and 15 as well. Currently, the updated indications for NOM include diagnostic / therapeutic angiography in carefully selected unstable trauma patients [bib_ref] Nonsurgical management of patients with blunt splenic injury: efficacy of transcatheter arterial..., Hagiwara [/bib_ref] [bib_ref] The role of interventional radiology in trauma, Zealley [/bib_ref]. A study by Wei showed that [bib_ref] Angioembolization reduces operative intervention for blunt splenic injury, Wei [/bib_ref] : - There were no patients with transient hypotension in which SAE failed -31% of patients who underwent SAE with good results were over 55 years old; - The spleen injury score was higher (3.8) than that of patients undergoing emergency surgery (3.4); - in 68% of the cases a distal SAE was performed with an average of 50% embolized splenic area; - reduced packed red cell / first 24 hours transfusion needs than operated patients; - radiological costs were similar to the surgical ones; - there were no differences concerning thrombo-embolic, pleural complications, mechanical ventilation-associated pneumonia and mortality rates between the 2 groups. The final decision depends on the experience of the trauma team involved and the technical possibilities of the trauma centers. Jeremitsky [bib_ref] Does splenic embolization and grade of splenic injury impact nonoperative management in..., Jeremitsky [/bib_ref] believes that SAE should be indicated in cases with active bleeding or in the presence of intrasplenic pseudoaneurysm (identified on CT scan), while splenic injury extent and the degree of hemoperitoneum are not absolute indications "per se". Wei [bib_ref] Angioembolization reduces operative intervention for blunt splenic injury, Wei [/bib_ref] considers that SAE is appropriate in severe injuries as well (grades IV or V) which associate severe haemoperitoneum (indication pointed out by Thompson as well- [bib_ref] Novel computed tomography scan scoring system predicts the need for intervention after..., Thompson [/bib_ref]. Imbrogno [bib_ref] Splenic artery embolization in blunt trauma. How I do it, Imbrogno [/bib_ref] believes that AES is indicated in hemodynamically stable patients in the following situations: -active extravasation of contrast; -severe splenic lesions (grade ≥ 3) and decreased hematocrit; -splenic vascular lesions associated with decreased hematocrit values. Changes in patients with normal indication of tomographic angiography but SAE are unclear. The most recent study on this topic [bib_ref] At first blush: Absence of computed tomography contrast extravasation in grade IV..., Bhullar [/bib_ref] provided the following information: -contrast blush on the initial CT examination; -grade IV-V splenic lesion on the initial CT examination; -decrease in Hb after admission, during NOM. The authors point out that, often, the indication of SAE involves the simultaneous presence of several factors mentioned in this paper. Their recommendation is conducting the angioembolization in all cases of splenic rupture, grades IV or V, regardless of the presence / absence of other findings and using it selectively for grade I-III injuries only if there is a contrast blush or declining Hb during NOM. The use of an aggressive SAE resulted in favorable NOM results in 80% of severe splenic injuries (grade IV-V) [bib_ref] Nonoperative management of blunt splenic injury: a 5-year experience, Haan [/bib_ref]. Howell's study [bib_ref] Delay to therapeutic interventional radiology postinjury: time is of the essence, Howell [/bib_ref] demonstrated that a delay in interventional radiology procedure initiation in hypotensive trauma patients with an average ISS of 17 is associated with a two-fold increase in risk of death. For each hour of delay the risk of death increases with 47%, regardless of the mechanism of injury. The faster the interventional radiology procedure is performed the better the results will be. The technical failure of SAE is defined as the inability to cannulate and embolize the splenic artery and its branches; the SAE is deemed as failed when subsequent surgery is needed to acquire hemostasis [bib_ref] The effects of splenic artery embolization on nonoperative management of blunt splenic..., Sabe [/bib_ref]. Thus, SAE failure is placed by this author around 5-6%. Selectively implementing SAE as nonoperative management for splenic trauma cases in patients at risk decreased the failure of this method to values of 2-4% [bib_ref] Blunt splenic injuries: nonsurgical treatment with CT, arteriography, and transcatheter arterial embolization..., Sclafani [/bib_ref] [bib_ref] Nonoperative salvage of computed tomographydiagnosed splenic injuries: utilization of angiography for triage..., Sclafani [/bib_ref] [bib_ref] Blunt splenic injuries: high nonoperative management rate can be achieved with selective..., Dent [/bib_ref] [bib_ref] Admission angiography for blunt splenic injury:advantages and pitfalls, Haan [/bib_ref]. Currently, the use of SAE resulted in a decreased number of splenic operations [bib_ref] Impact of splenic artery embolization on the success rate of nonoperative management..., Van Der Vlies [/bib_ref] , the frequency of emergency interventions performed have decreased from 33.3% to 11.9% after the introduction of this method [bib_ref] Management of blunt injuries to the spleen, Renzulli [/bib_ref]. The disadvantages of this method are: difficult monitoring and resuscitation (in Angiography suite), the need for an interventional radiologist, timeconsuming procedure (bearing the risk of a possible decompensation of the patient). Currently, there is a constant concern for the splenic functional changes post SAE. It is necessary to demonstrate that after angiographic maneuver, the residual splenic tissue is able to provide at least part of the previous functionality. In the absence of a normal immunocompetent spleen the risk of fulminant sepsis (OPSI) is permanent even if its frequency is below 1% (in multiple trauma adult patients). Harbrecht [bib_ref] Is anything new in adult blunt splenic trauma, Harbrecht [/bib_ref] concluded that the splenic immune function after SAE depends of the actual technique, blood vessel size and long-term development of collateral circulation. Similarly, Hagiwara [bib_ref] Nonsurgical management of patients with blunt splenic injury: efficacy of transcatheter arterial..., Hagiwara [/bib_ref] stresses the preservation of the splenic reticuloendothelial system's function post-embolization. Walusimbistudied the complete blood count and serum complement values and found no difference between patients with blunt spleen injuries that underwent SAE and those with blunt abdominal trauma without splenic involvement. Malhotra [bib_ref] Preservation of splenic immunocompetence after splenic artery angioembolization for blunt splenic injury, Malhotra [/bib_ref] concluded that after SAE for blunt splenic injuries, the immune function is partially or totally preserved. Nakae's [bib_ref] Does splenic preservation treatment (embolization, splenorrhaphy, and partial splenectomy) improve immunologic function..., Nakae [/bib_ref] retrospective study, however, proves that the splenic preservation (through embolization, splenorrhaphy, partial splenectomy) opposed to complete splenectomy does not provide significant advantages in terms of immune function, including IgM and 14 serotypes of anti-S. Pneumoniae antibodies. The authors strongly suggest implementing preventive measures and thorough follow-up. Post-embolization splenic immuno-competency remains questionable although there are studies that support its viability. However, current studies do not consider vaccination necessary after performing SAE for splenic trauma [bib_ref] Preserved splenic function after angioembolisation of high grade injury, Skattum [/bib_ref] [bib_ref] Nonoperative management and immune function after splenic injury, Skattum [/bib_ref]. SAE is an elegant solution for non-operative management of splenic trauma in all trauma centers. [fig] Figure 2: Grade IV splenic laceration -CT image. [/fig] [fig] Figure 3: Hemoperitoneum in Douglas pouch. [/fig] [fig] Figure 4: Splenic angiography -heterogeneous contrast uptake within the splenic parenchyma with fine areas of contrast extravasation. [/fig] [fig] Figure 5: Splenic angioembolization -postprocedural image. [/fig]

Severe obesity and diabetes insipidus in a patient with PCSK1 deficiency☆ Non-synonymous mutations affecting both alleles of PCSK1 (proprotein convertase 1/3) are associated with obesity and impaired prohormone processing. We report a proband who was compound heterozygous for a maternally inherited frameshift mutation and a paternally inherited 474kb deletion that encompasses PCSK1, representing a novel genetic mechanism underlying this phenotype. Although pro-vasopressin is not a known physiological substrate of PCSK1, the development of central diabetes insipidus in this proband suggests that PCSK1 deficiency can be associated with impaired osmoregulation. # Introduction Proprotein convertases (PCs) are a family of serine endoproteases that cleave inactive pro-peptides into biologically active peptides [bib_ref] The proprotein convertases, 20 years later, Seidah [/bib_ref]. Two family members, Proprotein Convertase Subtilisin/Kexin types 1 and 2 (PCSK1 and PCSK2) are selectively expressed in neuroendocrine tissues where they cleave a broad but specific set of prohormones including pro-opiomelanocortin (POMC), prothyrotrophin releasing hormone (TRH), proinsulin, proglucagon, and progonadotrophin releasing hormone (GnRH) [bib_ref] Processing of prothyrotropin-releasing hormone by the family of prohormone convertases, Schaner [/bib_ref] [bib_ref] The atypical 16p11.2 deletion: a not so atypical microdeletion syndrome?, Barge-Schaapveld [/bib_ref] [bib_ref] PC1 and PC2 are proprotein convertases capable of cleaving proopiomelanocortin at distinct..., Benjannet [/bib_ref] [bib_ref] Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic..., Dong [/bib_ref] [bib_ref] Attenuation of the polypeptide 7B2, prohormone convertase PC2, and vasopressin in the..., Gabreels [/bib_ref] [bib_ref] GAP-releasing enzyme is a member of the pro-hormone convertase family of precursor..., Rangaraju [/bib_ref] [bib_ref] Differential processing of proglucagon by the subtilisin-like prohormone convertases PC2 and PC3..., Rouille [/bib_ref] [bib_ref] Proinsulin processing by the subtilisin-related proprotein convertases furin, PC2, and PC3, Smeekens [/bib_ref]. Congenital deficiency of PCSK1 has previously been reported in three unrelated probands with severe hyperproinsulinemia, malabsorptive diarrhea, hypogonadotropic hypogonadism, partial central defects in the adrenal and thyroid axes and severe obesity [bib_ref] Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency, Jackson [/bib_ref] [bib_ref] Hyperphagia and early-onset obesity due to a novel homozygous missense mutation in..., Farooqi [/bib_ref]. At least some of these phenotypes can be explained by the known or suggested involvement of PCSK1 in the processing of proinsulin, proopiomelanocortin, proglucagon, proGnRH and proTRH [bib_ref] The proprotein convertases, 20 years later, Seidah [/bib_ref]. We describe the fourth patient with PCSK1 deficiency whose phenotype, in addition to the above, included central diabetes insipidus. ## Research design and methods Direct nucleotide sequencing of the PCSK1 gene was carried out as previously reported. SNP microarray analysis was performed using the Affymetrix 6.0 platform using 500 ng of total genomic DNA. Data was analyzed using Affymetrix Genotyping Console Browser v.3.01. Multiplex Ligation-independent Probe Amplification (MLPA) probes in the PCSK1 gene region (chr5:95751875-95774445) were designed following MRC-Holland (The Netherlands) recommendations (http://www.mlpa.com/) and sequences are available upon request. MLPA Hybridization, ligation and PCR were carried out using 200 ng of genomic DNA and the SALSA-MLPA kit (MRC-Holland, The Netherlands), according to the manufacturer's instructions. The MLPA PCR products (1 μl) were mixed with 0.5 μl GeneScan™-500 ROX™ size standard (Applied Biosystems, UK) and 10 μl of HiDi formamide (Applied Biosystems, UK) and separated on an ABI 3130 genetic analyzer (Applied Biosystems, UK) and electrophoresis data extracted using GeneMapper software v4.0 (Applied Biosystems, UK). # Results The male proband weighed 3.8 kg at birth. Diarrhea began at three days of life and persisted despite oral feeding with multiple formula changes. Endoscopy and flexible sigmoidoscopy revealed a grossly normal esophagus, stomach and duodenum, and scattered ulcers in the sigmoid colon. Pathology reported eosinophils present in the duodenum, stomach and colon and eosinophilic cryptitis in the colon consistent with "allergic colitis". Due to continued diarrhea and failure to thrive, parenteral nutrition together with continuous nasogastric feeds of an amino-acid based formula was instituted at 2 months of age. Watery diarrhea, however, continued. At 3 months the patient was admitted for sepsis associated with his intravenous catheter. Within 2 h of central line removal, his blood glucose fell to 33 mg/dl, with a low serum cortisol of 11.60 μg/dl (normal > 18 μg/dl) and a normal growth hormone (13.8 ng/ml). Thyroid function was consistent with central hypothyroidism (TSH 1.60 μIU/ml) with free thyroxine levels of 0.78 ng/dl (normal 0.9-1.8 ng/dl) and a low testosterone (28 ng/dl) and micropenis noted on clinical examination. In view of these findings, he was diagnosed with partial hypopituitarism and started on hydrocortisone and thyroxine replacement therapy. Monthly testosterone injections for 3 months resulted in the normalization of his penile size. Persistent polydipsia and polyuria were noted in the third year of life. Following restriction of fluid overnight, his serum Na was 145 mEq/l, Cl 107 mEq/l and osmolality was 304 mOsm/kg (urine specific gravity was 1.010 and osmolality 386 mOsm/kg), confirming the diagnosis of diabetes insipidus. He was started on oral desmopressin which led to a marked improvement in symptoms and electrolyte abnormalities. From 12 months, rapid weight gain began despite an apparent average caloric intake of~70 kcal/kg/day. Some solid foods were introduced for the first time at about 14 months, and this resulted in improved stool consistency and less frequent diarrhea. By 24 months old, the child was grossly obese . The amino-acid based formula was discontinued at 3 years of age. In view of the combination of chronic diarrhea, rapid weight gain and selective hypopituitarism, the possibility of PCSK1 deficiency was entertained. A nonfasting plasma insulin was low (0.2 μU/ml) but proinsulin was > 200 μU/ml. Direct nucleotide sequencing of PCSK1 revealed an apparently homozygous single base pair deletion which results in a frameshift after Pro 341, with the introduction of 91 abnormal amino acids terminating in a premature stop codon (W342GfsX92). The mutation is located within a region that encodes the catalytic domain and thus would be expected to abolish PCSK1 function. The proband's mother was found to be heterozygous for the same mutation. However, no PCSK1 mutation was found in his father. SNP microarray analysis revealed a 474kb interstitial deletion of 5q15-q15 (95669703-96143955) which contains three genes, PCSK1, calpastatin and endoplasmic reticulum aminopeptidase 1. This deletion was confirmed by Multiplex Ligation-independent Probe Amplification (MLPA) and was also found in the patient's father . Thus, the patient had inherited a large deletion that encompassed the PCSK1 gene and also a frameshift mutation within the gene resulting in the loss of both alleles and the phenotype of PCSK1 deficiency. Common heterozygous variants in PCSK1 have been associated with late-onset obesity [bib_ref] Common nonsynonymous variants in PCSK1 confer risk of obesity, Benzinou [/bib_ref]. However, both parents of this proband were of normal weight and had no evidence of intestinal or endocrine abnormalities. While these findings do not preclude a more subtle effect of some PCSK1 variants on body weight, the loss of both PCSK1 alleles appears to be necessary for the complete phenotype. # Discussion We describe the fourth patient with PCSK1 deficiency due to the combination of a maternally inherited frameshift mutation leading to a premature stop and a paternal deletion on chromosome 5 encompassing PCSK1 and two adjacent genes. This patient differs significantly from the three previous cases all of which involved homozygosity or compound heterozygosity for missense or splice site mutations [bib_ref] Small-intestinal dysfunction accompanies the complex endocrinopathy of human proprotein convertase 1 deficiency, Jackson [/bib_ref] [bib_ref] Hyperphagia and early-onset obesity due to a novel homozygous missense mutation in..., Farooqi [/bib_ref]. In this case one allele was deleted and the other disrupted by a 91 amino acid insertion followed by a premature stop codon occurring within the catalytic domain. While some residual PCSK1 enzymatic activity could conceivably have persisted in the three previously reported cases it seems highly likely that this patient represents the null phenotype of PCSK1. The development of central diabetes insipidus in our patient suggests that PCSK1 may be involved in the full functioning or central sensing of osmolality in humans. Whether this is due to the failure of provasopressin processing or to some other malfunction of osmoreception or vasopressin production or release is unknown. Vasopressin is synthesized in hypothalamic neurons expressing both PCSK1 and PCSK2 [bib_ref] Cellular localization of the prohormone convertases in the hypothalamic paraventricular and supraoptic..., Dong [/bib_ref]. In mice, both PCSK1 and PCSK2 are involved in the processing of pro-vasopressin to vasopressin, suggesting that a degree of redundancy may exist in vivo [bib_ref] Attenuation of the polypeptide 7B2, prohormone convertase PC2, and vasopressin in the..., Gabreels [/bib_ref] [bib_ref] Endocrinomic profile of neurointermediate lobe pituitary prohormone processing in PC1/3-and PC2-Null mice..., Hardiman [/bib_ref] [bib_ref] Analysis of peptides in prohormone convertase 1/3 null mouse brain using quantitative..., Wardman [/bib_ref]. In summary we report the fourth patient with PCSK1 deficiency. The finding of diabetes insipidus in this patient, and clinical features suggestive of this diagnosis in one patient previously reported by us [bib_ref] Hyperphagia and early-onset obesity due to a novel homozygous missense mutation in..., Farooqi [/bib_ref] , suggest that this potentially clinically significant and treatable abnormality should be sought in future patients presenting with this syndrome. . A. Growth chart of proband demonstrating marked failure to thrive over first 2 months; stabilization of weight gain from 2 months (with introduction of parenteral nutrition and an amino acid based formula) and excessive weight gain from 1 year. B. Family tree with chromatograms. Wildtype (Wt); deletion (del). C. MLPA confirmation of 474kb deletion of 5q15-q15 (95669703-96143955). Patient's MLPA traces are in red overlayed on the control MLPA traces in black. MLPA probes for genes in the region of interest are detailed below. Two of the MLPA probes are positioned within the PCSK1 gene and appear to be deleted in the patient (arrows), and two are on either side of the deleted region on chromosome 5q15 (distal and proximal probes), and they are identical to the normal control.

Randomised controlled trial using a theory-based m-health intervention to improve physical activity and sleep health in adults: the Synergy Study protocol For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtmlBMJ OpenSTUDY PROTOCOL 1Date: 03/08/2017; Version 1 2 3Title 4 5A randomised controlled trial using a theory-based m-health intervention to improve physical 6 activity and sleep health in adults: The Synergy Study protocol. 7 8ABSTRACT 1Introduction To decrease chronic disease rates, there is a need to reduce physical inactivity 2 and poor sleep health in the adult population. Given the high prevalence of these unhealthy 3 habits, behavioural interventions with potential for wide reach are needed. 4Methods and AnalysisThe aims of the Synergy Study will be to test the effect of a 3-month 5 personalised mobile app intervention on two main outcomes: minutes of moderate-to-6 vigorous physical activity and overall sleep quality. In addition, between-group changes in 7health-related quality of life and mental health status will be assessed as secondary outcomes. 8The pre-specified mediators and moderators include social cognitive factors, the 9 neighbourhood environment, health (BMI, depression, anxiety, stress) and sociodemographic 10 factors (age, gender, education). Assessments will be conducted after 3 months (primary 11 endpoint) and 6 months (follow-up). The intervention will provide access to a specifically 12 developed mobile app, through which participants can set goals for active minutes, daily step 13 counts, resistance training, sleep times and sleep hygiene practice. The app also allows 14 participants to log their behaviours daily and view progress bars as well as instant feedback in 15 relation to goals. The personalised support system will consist of weekly summary reports, 16 educational and instructional materials, prompts upon disengagement and weekly facts.Engaging in sufficient physical activity and maintaining good sleep health are two lifestyle 2 behaviours that significantly reduce the risk of all-cause mortality, 1 2 cardiovascular disease, 3 3 4 and type-2-diabetes. 5 6 Sufficient physical activity is the accumulation of at least 150 4 minutes of moderate-intensity or 75 minutes of vigorous-intensity physical activity per 5week. 7 Good sleep health is characterised by duration, quality and timing of sleep that leaves 6 a person satisfied with their sleep and alert during the day. 8 Internationally, up to 32% of 7 adults are insufficiently physically active, 9 and up to 29% report sleeping <6 hours, 10 24% 8 report poor quality sleep, 11 and >50% report inconsistent bed and wake times, the latter of 9 which are indicators of poor sleep health. 12 There is no global estimate of the percentage of 10 adults who report both, insufficient physical activity and poor sleep health. However, 11 evidence suggests that individuals with poor sleep health also report lower levels of physical 12 activity. 13 14 Thus, interventions which target both behaviours have the potential to make 13 meaningful contributions to public health. 14 15Multiple lifestyle behaviour interventions produce greater reductions in the risk of poor 16 health than interventions that target a single behaviour. 15 Moreover, physical activity and 17 sleep have a bidirectional relationship, 16 in which physical activity improves indicators of 18 sleep health (e.g., sleep quality) and good sleep health is associated with greater levels of 19 physical activity.17Interventions targeting both behaviours simultaneously may capitalise on 20 this reciprocal relationship to produce larger increases in both behaviours. 18 Previous reviews 21 of multiple behaviour interventions however, have not identified any studies that specifically 22 target changes in both physical activity and sleep health and tested the efficacy of this 23 approach in a randomised controlled trial.[19][20][21] frequently promote sleep hygiene, 22 using a set of self-regulatory strategies that help to 2 promote good sleep health, but details of behaviour change techniques (BCT) to support 3 changes in sleep hygiene behaviours, such as regular physical activity or stress management, 4 are usually not reported. [bib_ref] Effect of brief sleep hygiene education for 11 workers of an information..., Kakinuma [/bib_ref] Without providing the necessary guidance to promote behaviour 5 change, it is unlikely that such education-only interventions are likely to change behaviour, as 6 education-only interventions are known to be less effective than those that are combined 7 with additional self-regulation strategies. [bib_ref] Print versus website physical activity 14 programs: a randomized trial, Marshall [/bib_ref] Furthermore, multiple health behaviour change 8 interventions need to implement BCT that are specific to each behaviour to produce greater 9 changes in targeted behaviours. [bib_ref] Do single and multiple behavior change 17 interventions contain different behavior change..., Mc Sharry [/bib_ref] Interventions targeting physical activity and sleep in 10 combination therefore need to provide behaviour-specific intervention strategies to maximise 11 change and harness the potentially synergistic effects between physical activity and sleep. 12 [bib_ref] Sleep and society: an epidemiological perspective, Bixler [/bib_ref] Reviews of the evidence suggest theory-based interventions are more effective in changing 14 behaviour than interventions that do not use a theoretical approach. Theoretical models 15 provide important guidance for the development of behaviour change interventions, aiming 16 for the uniform operationalisation of cognitive and behavioural determinants. Cognitive Theory (SCT) is one of the most widely used theories in health behaviour 18 research. [bib_ref] Social cognitive theories used to 24 explain physical activity behavior in adolescents:..., Plotnikoff [/bib_ref] It aids the conceptual understanding of behaviour change, as it accounts for the 19 interactions between individual and environmental processes that either facilitate or impede 20 behaviour change . This is particularly relevant when targeting both, physical activity and 21 sleep health, since individual as well as environmental factors are known to influence both 22 behaviours. [bib_ref] Social cognitive theory and physical 33 activity: a systematic review and meta-analysis, Young [/bib_ref] SCT has guided the development of numerous physical activity interventions 23 and its constructs are strongly associated with physical activity. [bib_ref] Social cognitive theory and physical 33 activity: a systematic review and meta-analysis, Young [/bib_ref] [bib_ref] Social cognitive determinants of physical 36 activity in young adults: a prospective..., Rovniak [/bib_ref] However, there is limited 24 understanding of these social cognitive factors in relation to sleep health. - currently consuming hypnotics (sleep inducing medication); 3 - being employed in any night shift work; 4 - planning frequent travel (once a month or more often) to a destination with a shift in 5 time zone by more than three hours during the intervention period; 6 - currently using a self-monitoring system or device to track or log physical activity or 7 sleep (this includes non-device assisted applications); and 8 - not having access to an internet-enabled iOS (Apple) or Android smartphone or tablet. 9 10 Study procedure 11 Eligible participants will be contacted via Email and welcomed into the study. Participants 12 will be asked to complete online surveys assessing primary and secondary outcomes, 13 potential mediators/moderators and socio-demographics at three time points. [fig_ref] Figure 1: Social cognitive factors related to physical activity and sleep hygiene Flow of... [/fig_ref] illustrates the flow of participants throughout the trial. 15 [bib_ref] The bidirectional relationship between exercise and sleep: Implications for 32 exercise adherence..., Kline [/bib_ref] All online surveys will be administered using Qualtrics ® (Provo, Utah). If specified screening 17 criteria are not met, participants will be advised via text displayed at the end of their survey 18 and further contact will only be made where ambiguous responses require clarification. 19 Ineligible participants will also receive a link providing free and unlimited access to the 20 public version of the Balanced app. [bib_ref] Activity Trackers Implement Different Behavior 1 Change Techniques for Activity, Sleep, and..., Duncan [/bib_ref] ## 22 Participants will receive an Email with a unique password-protected link (URL) to their 23 survey at each assessment point. Each person who has completed their baseline survey will 24 be randomly allocated to one of two groups. Participants allocated to the intervention group 25 will be mailed a pedometer, tool sheets, login details and instructions for download and 1 installation of the "Balanced" smartphone app in the form of a participant handbook. The 2 initial "Balanced" app was specifically developed for scientific purposes and is described in 3 more detail elsewhere. [bib_ref] Activity Trackers Implement Different Behavior 1 Change Techniques for Activity, Sleep, and..., Duncan [/bib_ref] As part of the modifications to the previous app, the physical 4 activity component of the app was revised to include daily steps and resistance training in 5 addition to moderate-to-vigorous intensity physical activity; and sleep hygiene in addition to 6 sleep times and sleep quality. Regular app use is supported by an Email and text message-7 based support system (see [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] , which is initiated as soon as a participant has gained 8 access to the app. All messaging will follow a standardised protocol that was designed under 9 consideration of the specificity, timeliness and relevance of contents (see [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] , as those 10 are valued components in mobile apps designed to change health behaviours.Following 11 completion of their 3-month assessment, participants may continue to use the app as much or 12 as little as they like, but the message-based support will no longer be provided. The intervention is composed of app and non-app components, with non-app components 3 referring to any content of the intervention that is delivered via participant handbook, text 4 message or Email. 5 App components consist of educational resources, self-monitoring, goal-setting and feedback. 6 Participants will have continuous access to the app throughout the intervention period. For 7 the first 3 months, which is the time between baseline and the primary endpoint, these 8 components will be complemented by a messaging system providing personalised feedback 9 on progress towards goals, prompting goal review and prompting practice of the target 10 behaviours. The messaging component will cease at the 3-month assessment, but participants 11 will have continued access to the app. Following completion of the study, participants will be 12 able to continue to access and use the app for an indefinite period, however will not be 13 required to complete any further assessments as part of this study. The app will be available 14 on both, Android and Apple based operating systems. provides an overview of 15 intervention strategies used to operationalise the social cognitive constructs in the 16 intervention. [bib_ref] A review of lifestyle factors that contribute to 36 important pathways associated..., Lopresti [/bib_ref] 18 ## Educational resources 19 App resources will consist of educational information about the importance of the two 20 behaviours, basic instructions on how to change each behaviour and guidance for app use 21 (e.g., how to interpret traffic lights and progress graphs). Educational content will provide 22 participants with knowledge on the health benefits of each behaviour, the current national 23 guidelines for physical activity and sleep and the importance of resistance training and 24 incidental physical activity in addition to aerobic exercise, as well as the importance of all 25 recommendations based on summaries of the evidence. In addition to app content, 3 participants will receive a total of three tool sheets (enclosed in the handbook), one tool sheet 4 including goal-setting strategies, [bib_ref] Balanced: a randomised trial examining the 8 efficacy of two self-monitoring methods..., Duncan [/bib_ref] for each behaviour (one for physical activity, and one for 5 sleep), one that emphases action planning (again, one for each behaviour) and one tool sheet 6 with information and instructions adapted from publicly available resources for the practice 7 of stress management techniques (i.e., progressive muscle relaxation, deep breathing and 8 mindfulness). [bib_ref] Global health agenda on non-communicable diseases: has WHO set a smart 13..., Barreto Pds [/bib_ref] All tool sheets will be distributed along with the participant handbook at 9 outset (participants in the intervention group will receive their materials following 10 completion of their baseline assessment and waitlist controls will receive an identical 11 package following their 6-month assessment). In addition, during each month of the 12 intervention, one tool will be promoted (via Email) to encourage utilisation of these resources 13 (goal-setting at week 3, followed by action planning at week 6 and stress management at 14 week 9). Individuals are instructed to set goals that are personally relevant and meaningful to 15 them, but the goal-setting information provided will advise participants that their overarching 16 goal should be to gradually progress towards achieving the recommended minimum of 150 17 minutes of moderate-intensity physical activity per week, 7 and sleeping between 7 and 9 18 hours per night. Furthermore, participants will receive a weekly text message containing of 12 educational and motivational facts relating to physical activity and sleep for better 20 health (i.e., the consequences of poor sleep health). Each factoid message will also refer to 21 the resources section available in the app and encourage people to use it. Participants will be asked to recall minutes of moderate to vigorous physical activity, and 1 participation in resistance training, and manually enter this into the app every day. Daily 2 steps will be objectively measured using the pedometer (Yamax SW200) provided and 3 manually entered by participants into the app. Participants will not be asked to return their 4 pedometer. 5 . Operationalisation of social cognitive factors and behaviour change strategies SCT constructs BCT [bib_ref] Associations of objectively assessed physical activity 17 and sedentary time with all-cause..., Schmid [/bib_ref] Components Description of intervention components Participants will be asked to recall and enter their activity and sleep behaviours. The daily log will allow entries for active minutes, daily steps, resistance training sessions, sleep and wake times, a sleep quality rating, as well a checklist of 10 sleep hygiene goals. Participants will be asked to tick off those sleep hygiene goals they implemented the previous day. ## Self-efficacy Bar charts will provide a history for daily, weekly and 3-month progress in relation to goals per behaviour (for each of the items data are logged for). ## App dashboard traffic light The activity dashboard produces a traffic light colour relating to total active minutes, while the colour of the sleep dashboard relates to total sleep duration. Goals can be adjusted at any time, which will determine the colours on the dashboard traffic light. This is dynamically updated as soon as a self-monitoring entry is made: a green light indicates a participant is meeting, exceeding or close to their goal; an orange light indicates they are progressing toward their goal although are not close; and a red light indicates they are markedly below their goal. Tool sheets A series of tool sheets delivered at weeks 3, 6, and 9 will promote goal-setting and action planning and give detailed guidance on how to set SMART goals and follow through with an action plan in the face of barriers (i.e., by being prepared). ## Weekly summary (email) This support feature will provide an overview of weekly totals and averages per behaviour (if sufficient data are available) and prompt participants to review goals, if needed. ## Prompts (sms) If participants fail to log any data on more than 4 days per week, they will receive a message prompting them to resume logging. ## Behavioural capability Information on where and when to be active/engage in in sleep promoting behaviours Instructions on how to be active and engage in sleep promoting behaviours ## App resources The resources section will provide the current national guidelines on how much physical activity per week and how much sleep (hours) per night adults need. This section also includes brief content on the when, the where, who with and how of being active and sleeping well (e.g., sleep hygiene practices). ## Weekly facts (sms) Each week, participants will receive a short text message with educational content on activity and/or sleep and health to reinforce the importance of both behaviours. ## Tool sheets Tool sheets provide more detailed information that enable a person to make positive changes to their physical activity and sleep levels and include action plan templates and examples of exercises. These materials will also include stress management techniques, such as PMR and controlled breathing. ## Expectations/ expectancies Information about the behaviour in relation to health Tool sheets As part of the goal-setting tool sheet, participants will be asked to think about the reasons for wishing to improve their health behaviours and what they anticipate as personal benefits, following improved levels of activity and sleep (examples will be provided). ## App resources App log personal goals This section will include information on why activity and sleep are important and how they contribute to health and wellbeing. Participants will be asked to personalise their goals, but work towards recommended minima (150 MVPA/week; 7-9h sleep/night); goals are carried forward from previous entries unless adjusted ## Intentions/goals Goal-setting Action Planning Self-monitoring Prompt practice Time Management Teach use of prompts Time management ## App dashboard traffic light Tool sheets Participants will be encouraged to put equal effort into improving both PA and sleep. This means 2 amber lights are better than one green and one red light. Participants will receive goal-setting strategies and example action plans for guidance (per behaviour) as part of the tool sheets described above. One of 3 tools will be promoted specifically via Email at week 3, 6 and 9, respectively. ## Reminders Participants are advised to set a daily bedtime reminder (optional) on their phone, which is intended to prompt a person's bedtime routine and will promote regular bed times. App resources Environmental restructuring as part of good SH will be highlighted in the resource section and include details on how to manage the bedroom environment. Also includes information on activity & sleep in the social context and seeking support from those in the same household (housemates, partner, family members). ## Sociostructural factors (social support & environment) Use of prompts Environmental restructuring Barrier identification Plan social support Tool sheets This will include short examples on how to identify and manage barriers around being active and getting good sleep and how to utilise one's social support and environment in favour of activity and sleep. Note. [bib_ref] Associations of objectively assessed physical activity 17 and sedentary time with all-cause..., Schmid [/bib_ref] Behaviour changes techniques were specified in accordance with the 40-item taxonomy of behaviour change techniques by ; MVPA = moderate-tovigorous intensity physical activity; PA = physical activity; PMR = progressive muscle relaxation; SH = sleep hygiene; Self-monitoring of sleep in the app will also be manually entered by participants. The sleep 1 log consists of: bedtime (time of going to sleep), wake time (time of waking) and sleep 2 quality (rating scale from 0 to 5 where 5 indicates high sleep quality). As an additional 3 feature, this section of the app allows participants to log which sleep hygiene behaviours they 4 practiced the previous day [fig_ref] Figure 2: Sleep review only -http [/fig_ref]. These include consumption of caffeine, alcohol, 5 nicotine, excessive intake of fluids or heavy meals before bedtime, regulation of the impact 6 of light, noise and temperature in the bedroom, use of light-emitting devices, regular exercise, 7 maintenance of consistent sleep and wake times, having and following a bedtime routine, 8 creating comfort (e.g., proper pyjamas and bedding) and managing stress. Participants can 9 self-monitor these behaviours at any time of the day and update this information as many 10 times per day as they prefer. 11 12 ## Self-regulation 13 App feedback on behaviour will be provided using graphical displays of logged behaviour in 14 relation to the goals set by the participant [fig_ref] Figure 3: Screenshots of app screens for self-monitoring and feedback relative to goals 209x148mm [/fig_ref]. Two types of graphical feedback are 15 provided. There will be separate graphs for moderate-to-vigorous intensity physical activity, 16 steps, resistance training, sleep duration, sleep quality, sleep timing and sleep hygiene. This 17 information will provide a breakdown in the form of daily, 1-week and 3-month bar charts. 18 The second graphical feedback to participants is via the dashboard which changes to one of 19 three colours -green, orange and red in a traffic light system -to provide immediate feedback 20 on participants' behaviour to in relation their goals on a daily basis [fig_ref] Figure 3: Screenshots of app screens for self-monitoring and feedback relative to goals 209x148mm [/fig_ref]. The 21 comparison of actual behaviour to goals based on a percentage of the goal achieved allows 22 the use of consistent criteria across behaviours. This differs to the traffic light system 23 originally used in Balanced, since process data from that study alluded to participants 24 preferring to see this feedback based on goals rather than guidelines for each behaviour. [bib_ref] Activity Trackers Implement Different Behavior 1 Change Techniques for Activity, Sleep, and..., Duncan [/bib_ref] As part of the goal review strategies, participants will be encouraged to evaluate their 2 achievements in relation to goals and adjust their goals whenever needed. This will be 3 facilitated by a personalised weekly summary of the previous week, delivered via Email, so 4 that any reviews and adjustments of goals align with the most recent progress and foster self-5 efficacy. If a participant has logged data on less than 4 days per week (per behaviour), a text 6 message will be sent to prompt practice. 7 8 Waitlist control group 9 Upon enrolment and allocation, the waitlist control group will not receive any intervention 10 materials and only be required to complete their baseline, 3-month and 6-month assessments. 11 After the 6-month assessment is completed, participants in this group will receive full access 12 to the intervention. 13 ## 14 ## Randomisation 15 Participants will be randomly allocated to 2 groups (intervention or control) after having 16 completed their baseline assessment. Opaque sealed envelopes (n = 80 per group) will be 17 prepared by BM using permuted block randomization with block sizes of 4 and 8, following 18 the procedures suggested by Once a participant has completed their baseline 19 assessment, a researcher not associated with the study who is responsible for group allocation 20 will open the envelope that is next in sequence and inform the project leader about the 21 allocation outcome. Participants will be informed by the project leader and be sent a package 22 containing study materials (i.e., handbook and pedometer), if they have been allocated to the 23 intervention group (participants in the waitlist control group will receive their study materials 24 after completing their 6-month assessment). The only exception for contravention with the 25 F o r p e e r r e v i e w o n l y allocation sequence will be made if family members or couples living in the same household 1 enrol in the study, which would pose a high risk of contamination, especially between groups. 2 For this reason, all individuals who are identified as members of the same household will be 3 allocated to the same group. Neither the trial participants, nor the project lead (BM) will be 4 blinded to group assignment. 5 6 ## Outcome measures 7 All measures will be assessed via online survey at baseline, 3 months, and 6 months, except 8 for socio-demographics which will only be collected at baseline. The 3-month survey will 9 further include process evaluation items that measure system usability and participant 10 satisfaction (intervention group only). The two primary outcomes will be total minutes of 11 moderate-to-vigorous physical activity and sleep quality. To increase adherence to scheduled 12 assessments, participants who complete their survey will be entered into a draw for 1 of 5 $50 13 shopping vouchers. This information will not be provided prior to enrolment and is not 14 intended to function as an incentive for individuals to sign up to participate, but merely to 15 promote adherence to assessment requirements. [fig_ref] Table 3: Overview of outcome measures and assessment time points [/fig_ref] provides a summary of outcome 16 measures and assessment time points. All online surveys will be pilot-tested and locked prior 17 to study commencement to prevent any changes from being made once the study is 18 underway. All survey forms will be hosted on Qualtrics ® . 19 20 1 ## Physical activity 2 The Active Australia Questionnaire (AAQ has demonstrated acceptable reliability), 45 46 is 3 sensitive to change in interventions, [bib_ref] Randomization and allocation concealment: a practical guide for 34 researchers, Doig [/bib_ref] and provides a measure of both the frequency and 4 duration of moderate-and vigorous-intensity physical activity during the last week. This 5 includes the total time spent in recreational walking and transport, moderate-intensity 6 physical activity (e.g., swimming, golfing), aerobic activity (e.g., cycling, jogging) and 7 vigorous gardening or yard work. Total minutes of moderate-and vigorous-intensity physical 8 activity will be created by summing minutes of walking, moderate and vigorous (weighted by 9 2) intensity physical activity. 10 11 ## Sleep quality 12 The Pittsburgh Sleep Quality Index (PSQI) consists of 19 items and 7 component scores with 13 scores ranging from 0 to 21.Items refer to sleep disturbances over the last 30 days and 14 higher scores indicate poorer sleep quality and a score above 5 is commonly used to indicate 15 poor sleep quality. The current study will use the PSQI as a continuous score. The PSQI is 16 the most frequently used self-report instrument in sleep research. [bib_ref] The Pittsburgh Sleep Quality Index: a new 46 instrument for psychiatric practice..., Buysse [/bib_ref] The PSQI has 17 demonstrated good reliability (α = 0.83), is sensitive to change and has strong psychometric 18 properties. All of the 7 component scores (e.g., sleep efficiency and total sleep duration) 19 will be reported in addition to the total score. 20 21 ## Secondary outcomes 22 Health-related quality of life 23 Poor sleep quality and inadequate sleep duration are independently associated with low 24 health-related quality of life. 53 The RAND-12 is a reliable and widely used instrument that [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] physical and emotional problems, social functioning, emotional wellbeing, energy/fatigue, 2 pain as well as general perceptions of health. In addition to the RAND-12 scale, 3 additional 3 items that make up the energy/fatigue subscale in the 36-item version of the RAND will be 4 asked, so that this domain can be evaluated separately. This will allow improvements in 5 energy and fatigue during the course of the intervention to be assessed. 6 7 Depression, anxiety, and stress 8 The effect of changes in physical activity and sleep on participants' severity of depression, 9 anxiety and stress symptoms will be assessed using the Depression-Anxiety-Stress Scale 10 (DASS-21). The DASS-21 is reported to have satisfactory levels of internal consistency for 11 its total scale (r = 0.93) as well as for its individual scales for depression (r = 0.88), anxiety (r 12 = 0.82) and stress (r = 0.90). In addition, DASS-21 scores will be examined as a potential 13 moderator of intervention efficacy. 14 15 ## Resistance training 16 Since the AAQ does not capture resistance training and because the Synergy Study will 17 promote regular resistance training, the number and duration of resistance training sessions 18 per week will be assessed using two items adapted from previous studies that assessed 19 resistance training. 55 One item will ask participants: "In the last week, on how many days 20 have you participated in muscle strengthening activities (including weight/resistance 21 training)?" and "What do you estimate was the total time (in hours/minutes) that you spent 22 doing muscle strengthening activities (incl. weight/resistance training) in the last week?" The 23 original items were adapted by changing the recall period from the previous month to the week to align with the recall period used in the AAQ. 25 The Workforce Sitting Questionnaire (WFSQ) will provide a self-report measure of total 3 domain-specific sitting time (over the last week), on workdays and non-workdays. 56 Domains 4 include sitting time accumulated at work, watching TV, using a computer, using transport 5 and doing other leisure activities. The WFSQ captures sitting time across several domains 6 with acceptable validity (r = 0.45) and reliability (ICC = 0.63). Possible reductions in total 7 sitting time may be a result of increased amounts of time allocated to light/incidental or 8 moderate-to-vigorous physical activity. 57 9 10 ## Sleep timing 11 A modified version of the validated Sleep Timing Questionnaire will be used to assess the 12 variability in sleep and wake times on working days as well as non-working days. To 13 minimise participant burden, the instrument used will only include items on the stability of 14 usual bed and wake times, and the usual bed and wake times per se. Response options are 15 categorical and scored on a scale from 1-11 with lower scores indicating less variability in 16 bed or wake times (e.g., 1 = 0-15 min; 2 = 16-30 min; 11 = >4 hours). 17 18 ## Insomnia severity 19 The Insomnia Severity Index (ISI) is a valid and reliable instrument for measuring insomnia 20 severity. 59 It can be used to classify individuals as having no insomnia (0-7), sub-threshold 21 insomnia (8-14), moderate clinical insomnia [bib_ref] A Review of Multiple Health Behavior Change Interventions 28 for Primary Prevention, Prochaska [/bib_ref] [bib_ref] The bidirectional relationship between exercise and sleep: Implications for 32 exercise adherence..., Kline [/bib_ref] [bib_ref] A review of lifestyle factors that contribute to 36 important pathways associated..., Lopresti [/bib_ref] [bib_ref] Sleep and exercise: a reciprocal issue, Chennaoui [/bib_ref] [bib_ref] Comparative efficacy of simultaneous versus 43 sequential multiple health behavior change interventions..., James [/bib_ref] [bib_ref] Multiple Risk Behavior Interventions: Meta-analyses 1 of RCTs, King [/bib_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Daytime sleepiness is a component of sleep health. It will be measured using the Epworth 2 Sleepiness Scale (ESS), which assesses daytime sleepiness. This scale has demonstrated high 3 internal consistency (Crohnbach's alpha = 0.88) and good reliability (r = 0.82). 60 4 5 ## Process outcomes 6 Internet self-efficacy 7 Participants' confidence in using the smartphone app will be assessed using an adaptation of 8 the Internet Self-Efficacy Scale to capture participants' overall understanding of app 9 software, confidence in gathering information (educational resources) using the app and 10 learning to use the app, as well as the ability to troubleshoot and resolve app problems. 61 11 Participants will rate their agreement with a total of eight statements on a seven-point scale 12 from strongly disagree to strongly agree. [bib_ref] Sleep and society: an epidemiological perspective, Bixler [/bib_ref] 14 ## Perceived user satisfaction 15 The Cognitive-Affective Model of Perceived User Satisfaction (CAMPUS) will be used to 16 ask participants about thoughts and feelings associated with using the mobile app (Balanced). 17 A total of 23 items enquire about participant opinion on the effects and aesthetics as part of 18 the app design (15 items), its effectiveness and efficiency (5 items) and the level of 19 satisfaction experienced when using the app (3 items) with the following anchors: frustrated 20 -contented, unhappy -gratified and sad -joyful. Items will be adapted to refer specifically 21 to the Balanced app, for example "I would consider my experience with using the Balanced 22 app as innovative". This instrument has demonstrated adequate levels of reliability and 23 validity. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Overall interaction with the app will be measured continuously throughout the study period 2 by the app database, which records the time and date a self-monitoring entry was made and 3 the actual value or response entered into the app. Analysis of usage patterns will include the 4 number of self-monitoring entries made and the duration of self-monitoring throughout the 5 intervention, similar to previous research. 63 64 These data will also be considered as a 6 mediator of behaviour change in the intervention group. 7 8 Usability of the App 9 App usability will be assessed using the 10-item System Usability Scale, 65 a valid and 10 reliable tool that assesses participant satisfaction relating to the utility of the websites on a 5-11 point scale (items will be reworded for smartphone app usability). Higher total scores (range 12 1-100) relate to better usability. 13 ## 14 ## Utility, advice acceptability and relevance of the app 15 A participant sub-sample (10%) will be determined by random selection for semi-structured 16 telephone interviews, which will take place once all participants have completed their 6-17 month assessments. These interviews will contribute valuable information for process 18 evaluation and include general personal feedback, desirable improvements and preferences 19 relating to future use. As part of these interviews, participants will be asked about their 20 perception of the app's usefulness to improve changes in self-efficacy levels (confidence) 21 toward physical activity and sleep health, coping with potential impediments (barriers) to 22 being more active and sleeping better, maintaining new routines/action plans and keeping it a 23 priority to be more active and sleeping better. Finally, advice acceptability and relevance in 24 terms of the content will be examined based on a previously used questionnaire. [bib_ref] A new method for measuring daytime sleepiness: the Epworth sleepiness 1 scale, Johns [/bib_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] 1 ## Mediators and moderators 2 ## Social cognitive factors 3 The testing of social cognitive factors as potential mediators of intervention efficacy may 4 provide insights into some of the underlying mechanisms involved in behaviour change, as 5 observed in previous behaviour change interventions. [bib_ref] Coursaris CK, van Osch W. A Cognitive-Affective Model of Perceived User Satisfaction..., Eastin [/bib_ref] Constructs from Social Cognitive 6 Theory will be assessed based on items from previously developed scales, with separate 7 items for each of the two behaviours relating to the person's projections towards their 8 occurrence over the next three months. For each behaviour, items (as adapted from previous 9 studies) will be probing the constructs of self-efficacy, [bib_ref] A new method for measuring daytime sleepiness: the Epworth sleepiness 1 scale, Johns [/bib_ref] intentions/goals, [bib_ref] Testing mediator variables in a physical 25 activity intervention for women with..., Plotnikoff [/bib_ref] and action planning. [bib_ref] Testing mediator variables in a physical 25 activity intervention for women with..., Plotnikoff [/bib_ref] [fig_ref] Table 3: Overview of outcome measures and assessment time points [/fig_ref] summarises, for physical activity and sleep 12 hygiene behaviours, the number of items per construct and the response options for each item 13 (See [fig_ref] Table 4: Social cognitive factors related to physical activity and sleep hygiene behaviours [/fig_ref]. 14 15 For physical activity, a total of 34 items will be used to assess the social cognitive factors. 16 Items will be framed around the statement "Regular physical activity is defined as doing at 17 least 150 minutes of moderate intensity physical activity each week. Moderate intensity can 18 be described as any type of aerobic activity performed at a level where a person begins to 19 lightly sweat, but can still carry on a conversation. This may feel different from one person to 20 another." These items will be scored by using the sum of items per construct. For sleep, a 21 total of 72 items will be used to assess the eight social cognitive factors. Each social 22 cognitive item will be based on each of the following nine sleep hygiene behaviours: (1) 23 avoiding caffeinated beverages (coffee, tea, energy drinks, etc.) in the late afternoon or right 24 before bedtime, (2) avoiding nicotine right before bedtime, (3) avoiding alcohol right before 25 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] bedtime, (4) exercising regularly, (5) reducing stress levels, (6) reducing the impact of noise 1 and nuisance in the bedroom, (7) keeping sleep and wake times consistent, (8) avoiding 2 daytime naps and (9) avoiding the use of technological devices (e.g., phone, TV, laptop, etc.) 3 right before bedtime or in bed. To avoid overburdening participants, each construct will be 4 assessed using a single item per sleep hygiene behaviour (nine in total). Thus, each social 5 cognitive construct will have nine items. For example, the self-efficacy construct will be 6 assessed by participants indicating their self-efficacy to perform each of the nine sleep 7 hygiene behaviours. Each construct will be scored as the sum of the nine sleep hygiene items; 8 however, the environment construct will not be included for sleep hygiene behaviours, as this 9 is already captured as part of the perceived neighbourhood disorder questionnaire. 10 11 (1) strongly disagree (5) strongly agree Social support 9 (1) strongly disagree (5) strongly agree Implementation intentions 9 (1) no, not really (7) strongly intend and Action planning 9 (1) no detailed plans (7) detailed plans Note. Each item per construct will refer to one of nine different sleep hygiene behaviours 1 Automaticity 2 Habits relating to lifestyle behaviours are non-conscious processes, which can act as 3 determinants of behaviour and may even regulate behaviour independently of changes in 4 conscious processes such as intention. The role that behavioural automaticity plays in the 5 context of physical activity and sleep behaviours, respectively, will be taken into account 6 using 1 item from the Automaticity Index per sleep hygiene behaviour (9 items), 75 and all 4 7 items of the index relating to physical activity (13 items in total), for example: "Reducing the 8 impact of noise in my bedroom is something I do automatically.", "Exercise is something I 9 do without thinking.". 10 11 ## Sleep hygiene 12 Sleep hygiene will be assessed to measure changes in in sleep hygiene behaviour using the Perceptions about the order or disorder within a person's physical and/or social environment 7 (i.e., neighbourhood peacefulness, safety, cleanliness) can have a significant influence on 8 physical activity levels and the quality and duration of sleep. 77-79 A person's neighbourhood 9 environment can also negatively affect mental health and participation in other health 10 behaviours. Based on an existing scale of neighbourhood disorder, which demonstrated 11 good levels of construct validity and internal consistency/reliability, 81 4 items will assess 12 each of the following characteristics of neighbourhood disorder: physical disorder and 13 physical order, social disorder and social order. These are assessed using the following 14 items: (1) "My neighbourhood is noisy", (2) "My neighbourhood is clean", (3), "There is a 15 lot of crime in my neighbourhood" and (4) "My neighbourhood is safe" These items will be 16 answered on a 5-point scale from strongly disagree (1) to strongly agree (5) and the average 17 responses across the four items will be calculated. 18 ## 19 ## Sample characteristics 20 A range of demographic and socioeconomic factors such as age, gender, height and weight, 21 education, income, marital status, occupation, working hours, etc. will be assessed. 22 Participants will be asked to also indicate (allowing multiple selection) whether they have 23 been told by a doctor that they have any of the following chronic diseases: arthritis, asthma, 24 cancer, cerebrovascular disease (stroke), chronic obstructive pulmonary disease 25 (emphysema), coronary heart disease (heart attack, angina), type-1 diabetes, type-2 diabetes, 1 high blood pressure, kidney disease, mental illness (depression, anxiety, etc.), osteoporosis, 2 irritable bowel syndrome, high cholesterol, or any other disease (to be specified by the 3 participant). In addition, participants will be assessed for morningness or eveningness type, 82 4 as eveningness types are thought to be more prone to engage in activities that cause social 5 jetlag, due to misalignments between times of sleep and times of social activity. 83 6 7 Power and sample size 8 Meta-analyses of physical activity interventions typically report small to moderate increases 9 in physical activity (Cohen's d = 0.14 -0.68). Moreover, poor sleep health, specifically 10 the duration or quality of sleep, has small to moderate magnitude associations with lower 11 physical activity levels. [bib_ref] Psychosocial and environmental factors associated with 18 physical activity among city dwellers..., Duncan [/bib_ref] Meta-analyses of non-pharmacological sleep interventions report 12 small to medium effect sizes for changes in sleep quality (Hedge's g = 0.35 and Cohen's d = 13 0.41) in clinical populations, and medium to large effect sizes (d = 0.74) in studies using 14 exercise to improve sleep. [bib_ref] Neighborhood disorder, psychophysiological distress, and 30 health, Hill [/bib_ref] Therefore, based on these observations and feasibility data from 15 the combined intervention, [bib_ref] Activity Trackers Implement Different Behavior 1 Change Techniques for Activity, Sleep, and..., Duncan [/bib_ref] it was assumed that a 3-month combined physical activity and 16 sleep intervention that specifically targets both behaviours and leverages the bi-directional 17 relationship between behaviours is likely to produce moderate increases in physical activity 18 Meta-analyses of physical activity and sleep interventions report average drop-out rates of 4 20%, however the majority of web-based trials report drop-out rates that are higher than 5 that, 84 thus the sample size for this study will be inflated to account for a 25% drop-out in the 6 current study. Therefore, the total sample size is 80 participants per group or 160 in total. A 7 sample of this size will also be adequately powered to detect mediated effect sizes of small (β 8 = 0.14) magnitude. [bib_ref] Does sleep quality mediate the association between 33 neighborhood disorder and self-rated..., Hale [/bib_ref] The participant recruitment phase will conclude once 160 complete 9 baseline responses have been obtained. 10 11 ## Analyses 12 Analyses will apply the intention-to-treat principle. Analysis of primary outcomes will be 13 blinded to group allocation and overseen by an independent statistician. The primary aim of 14 this study will be to examine differences in physical activity and sleep quality between the 15 intervention group and the control group at the 3-month primary time point. Between-group 16 differences in physical activity (AAQ minutes) and sleep quality (PSQI) will be estimated 17 using Generalised Linear Mixed Models (GLMM) adjusting for baseline measures of the 18 outcome, including all available data in the analysis. The model will include fixed effects for 19 group, time and their interaction. A random intercept will be used to account for repeated 20 measures on individuals. Separate GLMM will be used to examine changes in physical 21 activity and sleep. 22 ## 23 Sensitivity analyses using Pattern Mixture Modelling will be conducted to examine the 24 impact of missing data on outcomes. Where the GLMM assumes data are missing at random, 25 Pattern Mixture Modelling is robust to the assumed pattern of missing data. Group 1 differences in secondary outcome measures will be estimated using the same linear mixed 2 modelling approach, setting an alpha of 0.025 for each outcome. Potential mediators and 3 moderators of intervention efficacy will be examined using established approaches. Generalised linear mixed models and survival analysis will be used to examine differences in 5 usage patterns. 6 7 Data monitoring 8 Survey data will be exported directly from Qualtrics ® as a text file and imported into Stata for 9 scoring and analysis. A detailed database will track participants' progress through the trial 10 including the scheduling of assessments and reminders to complete assessments. Intervention 11 usage will be monitored throughout the trial by BM and MJD by way of the password-12 protected app database. Given the purpose of the trial, the data to be collected as well as the 13 nature of the intervention, no Data Monitoring Committee will be established. Detailed 14 strategies, including Email/text message reminders will be used to remind participants about 15 upcoming assessments. All Newcastle-based members of the research team (BM, MJD, ATR, 16 RCP) and other associated personnel will have access to the information in both identified 17 and re-identifiable forms. Should statistical analysis advice be sought, access to the data will 18 be granted in re-identifiable form using unique numerical identifiers and access approved by 19 the relevant Ethics Committee. 20 21 Print data will be stored in locked filing cabinets accessible only to the research team. 22 Electronic data will be stored on password-protected computers or servers only accessible to 23 the research team. Data will be retained for [bib_ref] A Review of Multiple Health Behavior Change Interventions 28 for Primary Prevention, Prochaska [/bib_ref] Results from the outcome measures will not be presented in a way that adversely affects the 4 confidentiality of participants. The description of participants will not allow identification of 5 individual participants, and individual results and individual names will not be revealed. 6 Final reports and publications will only consist of aggregated results. At the completion of 7 the study, participants will receive a plain English summary of study results. ## Declaration of interests 23 The authors and principal investigators of this study protocol declare no conflict of interest. Any amendments to the study protocol will be submitted to the Human Research Ethics 2 Committee (HREC) and updated on the trial register (ANZCTR) once full ethical approval 3 has been obtained. All authors meet ICMJE criteria for authorship in that they have 4 contributed substantially to the conceptual design; or the processes of data collection, 5 analysis or interpretation; the drafts and revisions of the study protocol and manuscript; 6 granted approval of the final version of the study protocol; and acknowledged their 7 accountability with regards to the integrity and accuracy of this study protocol. [bib_ref] Social jetlag: misalignment of biological and 39 social time, Wittmann [/bib_ref] In detail, the 8 first author of this protocol (BM) will be responsible for administrative and managerial 9 procedures related to all phases of the trial, which will be supervised by MJD and RCP. ATR Any type of adverse events reported by study participants, whether they be due to their 17 participation or due to an unrelated cause will be recorded and reported to the HREC. This 18 may include events reported by participants, including musculoskeletal injuries associated 19 with the uptake or increase in physical activity or emotional distress due to any survey items 20 of sensitive nature. Great care will be taken to avoid and prevent adverse events and the 21 research team will provide every possible assistance to remediate those events, should they 22 occur. The participant information statement interested individuals will have access to prior 23 to consenting to participate details any potential risks of discomfort associated with 24 It is advised that adults accumulate a weekly minimum of 150 minutes of moderate intensity 5 physical activity, combined with muscle strengthening activities on two days per week, 7 and 6 also achieve 7-9 hours of sleep per night. [bib_ref] Meta-analysis of internet-delivered 42 interventions to increase physical activity levels, Davies [/bib_ref] Achieving the recommended levels of physical 7 activity and sleep contribute and maintain optimal health and wellbeing through risk 8 reductions associated with chronic diseases such as heart disease and type-2-diabetes. Engaging in optimal levels of physical activity and sleep health can also positively contribute 10 to long-term weight management, mental health and overall quality of life. Notwithstanding this wide spectrum of benefits, a large proportion of the population does not 12 accumulate sufficient physical activity and/or achieve optimal sleep. 13 ## 14 Wide reaching behavioural programmes, such as those offered through m-health 15 interventions, have the potential to elicit the much needed shift of relatively large groups of 16 the population toward levels of physical activity and sleep that meet recommendations. [bib_ref] Computerised cognitive behavioural therapy for insomnia: a 4 systematic review and meta-analysis, Cheng [/bib_ref] To 17 yield positive changes in health behaviour, such interventions need to include educational 18 information, incorporate behaviour change techniques and deliver a level of guidance that 19 endorse the initiation and maintenance of health behaviour change. 29 98 Systematic reviews of 20 the effectiveness of multiple health behaviour interventions suggested that those targeting 21 related behaviours (e.g., diet and physical activity) produced greater behaviour change than 22 those targeting unrelated behaviours (e.g., smoking and physical activity),and that specific 23 intervention techniques are necessary for each behaviour. [bib_ref] Do single and multiple behavior change 17 interventions contain different behavior change..., Mc Sharry [/bib_ref] Physical activity and sleep are 24 suggested to have a bi-directional relationship, [bib_ref] The bidirectional relationship between exercise and sleep: Implications for 32 exercise adherence..., Kline [/bib_ref] relationship between physical activity and sleep. The Synergy Study addresses this by 2 targeting both physical activity and sleep, simultaneously, using specific intervention 3 techniques to enhance participants' self-regulatory skills in relation to the two health 4 behaviours and thus, leverages the potential for synergistic improvements in both behaviours. 5 An advantage of this study lies in its mode of delivery, which involves mobile technology 6 and therefore blends into day-to-day life. A key intervention strategy is the use of goal-setting 7 and feedback to promote behaviour change. It seeks to achieve this through the promotion of 8 dynamic goals and action plans, the implementation of a personalised support system further 9 addresses potential barriers (i.e., low levels of self-efficacy) that can increase the gap 10 between participant intentions and behavioural outcomes, [bib_ref] A general approach to causal mediation analysis, Imai [/bib_ref] and contribute to long-term 11 behaviour maintenance. This includes knowledge on how to set attainable goals and having 12 strategies in place that facilitate the occurrence of healthy behaviours, despite challenging 13 situations or unfavourable environmental factors. The structured promotion of goal-setting 14 strategies, combined with action plans that define in detail how an individual will implement 15 the intended behaviour, is known to be effective in changing health behaviours. Additional strengths of this study include its randomised waitlist controlled study design, 18 which will allow making inferences about the causal links between the intervention and 19 changes in behaviour, including participants from the wider public. While it is the first aim of 20 this study to test the intervention's efficacy to produce changes in two primary outcomes, the 21 pre-specified secondary outcomes (mental health, quality of life) will give insight into 22 changes in parameters of health and wellbeing that may be very meaningful to the participant. 23 And finally, this study will generate knowledge on social cognitive determinants of behaviour 24 change relating to sleep health and explore how these factors differ between physical activity 25 successful behaviour change in both behaviours and also further the application of social 2 cognitive theories in the multi health behaviour context. The limitations of this study include 3 the study duration, which, although at 6 months is longer than many studies, [bib_ref] Towards parsimony in habit measurement: testing 11 the convergent and predictive validity..., Gardner [/bib_ref] does not 4 provide insight into longer term changes and behaviour maintenance; and the lack of a 5 comparator condition receiving only the sleep or the physical activity programme to 6 determine the magnitude each intervention component has on its own. It is beyond the scope 7 of this study to test long-term efficacy exceeding 6 months, but future trials may be 8 encouraged to do this, provided the Synergy Study proves efficacious in the short term with 9 indications of effect retention at the 6-month time point. 10 ## 11 # Conclusion 12 This study protocol provides the rationale and methods associated with the implementation 13 and evaluation of the Synergy Study, a theory-based m-health intervention including 14 personalised support, with the aim to improve physical activity and sleep health in adults. To the authors' knowledge, this study will be the first to simultaneously target 16 changes in these two behaviours, using a sophisticated combination of technologies and 17 evidence-based strategies and test the efficacy of this approach in a randomised controlled 18 trial. Findings from this trial will provide valuable knowledge pertaining to the design of m-19 health interventions that combine behaviours in a format with wide reach. 20 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] ## 21 ## Legend of tables 22 ## Scientific title A randomised controlled trial using a theory-based m-health intervention to improve physical activity and sleep health in adults: The Synergy Study protocol. ## Countries of recruitment australia ## Health condition(s) or problem(s) studied Physical inactivity and poor sleep health will be targeted to promote the prevention of chronic diseases such as type-2 diabetes, heart disease and obesity through greater levels of physical activity and better sleep health (characterised by optimal sleep duration, good sleep quality and consistent bed and wake times). If the study is conducted in healthy human volunteers belonging to the target population ## Intervention(s) Intervention Group: The intervention group will receive a combined physical activity and sleep intervention using a mobile app and personalised support for 3 months Intervention Description: The mobile app will operationalise goal-setting, self-monitoring and feedback strategies in relation to physical activity and sleep health. The messagebased support service (Email and text messaging) will provide feedback on progress towards goals, prompts to practice the two behaviours and review/adjust goals, daily reminders (optional) and tool sheets on goal-setting, action planning and stress management. Participants will be asked to self-monitor their daily physical activity and sleep behaviours and set personal goals for both behaviours within the app. We will advise participants to log both behaviours on a daily basis, whereas goals can be reset at any time (if need be) or otherwise will remain unchanged. A summary report, which is sent to participants once weekly will assist with the process of goal review. Goal achievement is considered successful, if participants are within 20% of their goal. For example, if a participant aimed for 30 min of activity, their feedback dashboard (traffic light) will display a green light and their goal is reached, if the participant logged at least 24 min of activity or more for that day. Multiple entries per day are possible. A pedometer will be shipped to participants to facilitate the logging of a daily step count. Participants will be asked to set relevant and achievable goals (guidance provided); however, we will encourage individuals to gradually work towards the recommended minimum of physical activity (PA) and sleep as per national guidelines, which is 150 min/week (plus 2 days of resistance training) for PA and 7-9 hours of sleep per night. The app and all other components are informed by social cognitive theories and follow behaviour change taxonomies to ensure consistency with implementation standards and reporting. Participants will be given continuous access to the app. Waitlist Control Group: Following the 6-month follow-up assessment, thee waitlist control group will receive full access to the intervention Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recommended (see 8 Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any statistical methods to handle missing data (eg, multiple imputation) 30 ## Key inclusion and exclusion criteria # Methods: monitoring Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed 31 21b Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial n/a Introduction To decrease chronic disease rates, there is a need to reduce physical inactivity 2 and poor sleep health in the adult population. Given the high prevalence of these unhealthy 3 habits, behavioural interventions with potential for wide reach are needed. 4 # Methods and analysis The aims of this 2-arm RCT will be to test the effect of a 3-month 5 personalised mobile app intervention on two main outcomes: minutes of moderate-to-6 vigorous physical activity and overall sleep quality. In addition, between-group changes in 7 health-related quality of life and mental health status will be assessed as secondary outcomes. 8 The pre-specified mediators and moderators include social cognitive factors, the 9 neighbourhood environment, health (BMI, depression, anxiety, stress) and sociodemographic 10 factors (age, gender, education). Assessments will be conducted after 3 months (primary 11 endpoint) and 6 months (follow-up). The intervention will provide access to a specifically 12 developed mobile app, through which participants can set goals for active minutes, daily step 13 counts, resistance training, sleep times and sleep hygiene practice. The app also allows 14 participants to log their behaviours daily and view progress bars as well as instant feedback in 15 relation to goals. The personalised support system will consist of weekly summary reports, 16 educational and instructional materials, prompts upon disengagement and weekly facts. 17 ## Ethics and dissemination the human research ethics committee of the university of 18 Newcastle, Australia granted full approval: H-2016-0181. This study will assess the efficacy 19 of a combined behaviour intervention, mechanisms of behaviour change and gather high-20 quality process data, all of which will help refine future trials. Dissemination of findings will 21 include publication in a peer-reviewed journal and presentation at national or international 22 conferences. Participants will receive a plain English summary report of results. 23 Strengths and limitations of this study 4 ## Trial registration number actrn12617000376347 24 - No previous studies have tested the efficacy of a mobile intervention targeting 5 physical activity and sleep in combination in a randomised waitlist-controlled trial in 6 physically inactive adults reporting poor quality sleep, who do not have a sleep 7 disorder. 8 - Outcome data will contribute to the knowledge relating to chronic disease prevention 9 through multi health behaviours using a mobile intervention with wide reach. 10 - Findings will facilitate the future design of technology-based multi health behaviour 11 change interventions. 12 - Limitations include the lack of an intervention arm which receives only the physical 13 activity or only the sleep intervention. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Engaging in sufficient physical activity and maintaining good sleep health are two lifestyle 2 behaviours that significantly reduce the risk of all-cause mortality, 1 2 cardiovascular disease, and type-2-diabetes. 5 6 Sufficient physical activity is the accumulation of at least 150 4 minutes of moderate-intensity or 75 minutes of vigorous-intensity physical activity per 5 week. 7 Good sleep health is characterised by duration, quality and timing of sleep that leaves 6 a person satisfied with their sleep and alert during the day. Internationally, up to 32% of 7 adults are insufficiently physically active, 9 and up to 29% report sleeping <6 hours, 10 24% 8 report poor quality sleep, [bib_ref] Does nighttime exercise really disturb 14 sleep? Results from the 2013 National..., Buman [/bib_ref] and >50% report inconsistent bed and wake times, the latter of 9 which are indicators of poor sleep health. [bib_ref] Greater bed-and wake-time variability is 18 associated with less healthy lifestyle behaviors:..., Duncan [/bib_ref] There is no global estimate of the percentage of 10 adults who report both insufficient physical activity and poor sleep health. However, 11 evidence suggests that individuals with poor sleep health also report lower levels of physical 12 activity. Thus, interventions which target both behaviours have the potential to make 13 meaningful contributions to public health. 14 15 Multiple lifestyle behaviour interventions produce greater reductions in the risk of poor 16 health than interventions that target a single behaviour. [bib_ref] A Review of Multiple Health Behavior Change Interventions 28 for Primary Prevention, Prochaska [/bib_ref] Moreover, physical activity and 17 sleep have a bidirectional relationship, [bib_ref] The bidirectional relationship between exercise and sleep: Implications for 32 exercise adherence..., Kline [/bib_ref] in which physical activity improves indicators of 18 sleep health (e.g., sleep quality) and good sleep health is associated with greater levels of 19 physical activity. [bib_ref] A review of lifestyle factors that contribute to 36 important pathways associated..., Lopresti [/bib_ref] Interventions targeting both behaviours simultaneously may capitalise on 20 this reciprocal relationship to produce larger increases in both behaviours. [bib_ref] Sleep and exercise: a reciprocal issue, Chennaoui [/bib_ref] Previous reviews 21 of multiple behaviour interventions however, have not identified any studies that specifically 22 target changes in both physical activity and sleep health and tested the efficacy of this 23 approach in a randomised controlled trial. [bib_ref] Comparative efficacy of simultaneous versus 43 sequential multiple health behavior change interventions..., James [/bib_ref] frequently promote sleep hygiene, 22 using a set of self-regulatory strategies that help to 2 promote good sleep health, but details of behaviour change techniques (BCT) to support 3 changes in sleep hygiene behaviours, such as regular physical activity or stress management, 4 are usually not reported. Without providing the necessary guidance to promote behaviour 5 change, it is unlikely that such education-only interventions are likely to change behaviour, as 6 education-only interventions are known to be less effective than those that are combined 7 with additional self-regulation strategies. [bib_ref] Print versus website physical activity 14 programs: a randomized trial, Marshall [/bib_ref] Furthermore, multiple health behaviour change 8 interventions need to implement BCT that are specific to each behaviour to produce greater 9 changes in targeted behaviours. [bib_ref] Do single and multiple behavior change 17 interventions contain different behavior change..., Mc Sharry [/bib_ref] Interventions targeting physical activity and sleep in 10 combination therefore need to provide behaviour-specific intervention strategies to maximise 11 change and harness the potentially synergistic effects between physical activity and sleep. and its constructs are strongly associated with physical activity, but there is only limited 24 understanding of social cognitive factors in relation to sleep health. However, it may be 25 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] ## Study design 2 A two-arm randomised controlled (superiority) trial with a combined physical activity and 3 sleep intervention and a waitlist control group, with assessments conducted at 0 (baseline), months (primary endpoint) and 6 months (follow-up). 5 6 Recruitment 7 Digital and print-based advertising will be used to recruit nationwide in Australia. 8 Recruitment for both intervention arms commenced in May 2017 and will conclude once 9 sample size requirements are achieved (n = 160, refer to power and sample size section). 10 Social media advertising will be used to recruit in social media networks (e.g., Twitter, 11 Facebook) using target audiences that match inclusion criteria (i.e., age, living in Australia). 12 Electronic and print-based advertising will include magazines and newspapers with state-13 wide reach. All recruitment materials will provide contact details and a link to the consent 14 form and eligibility survey. Due to the remote delivery of the intervention in combination 15 with self-report based assessments, participants will not be required to visit the research 16 centre. [bib_ref] A review of lifestyle factors that contribute to 36 important pathways associated..., Lopresti [/bib_ref] 18 ## Exclusion criteria 19 Individuals who meet any of the following criteria will not be eligible to participate: 20 - not residing in Australia; 21 - not being between 18 and 55 years old; 22 - reporting a height and weight that is not consistent with a BMI between 18.5 and 35; 23 - accumulating more than 90 minutes of moderate/vigorous physical activity per week; 24 - rating their sleep-quality (over the past month) as fairly good or very good; 25 - having a condition that would make it unsafe or limits their ability to increase activity 2 levels or change sleep behaviours; 3 - having a diagnosed sleep disorder (chronic insomnia, sleep apnoea, sleepwalking, 4 narcolepsy, restless legs syndrome, etc.); 5 - currently consuming hypnotics (sleep inducing medication); 6 - being employed in any night shift work; 7 - planning frequent travel (once a month or more often) to a destination with a shift in 8 time zone by more than three hours during the intervention period; 9 - currently using a self-monitoring system or device to track or log physical activity or 10 sleep (this includes non-device assisted applications); and 11 - not having access to an internet-enabled iOS (Apple) or Android smartphone or tablet. 12 [bib_ref] Sleep and society: an epidemiological perspective, Bixler [/bib_ref] Interested participants who indicated already using a self-monitoring system or tracking 14 device were excluded to avoid the potentially confounding effect that the use of a self-15 monitoring system or device may have on behaviour, as most popular health apps or the 16 trackers themselves frequently implement a variety of behaviour change strategies. [bib_ref] Activity Trackers Implement Different Behavior 1 Change Techniques for Activity, Sleep, and..., Duncan [/bib_ref] 18 ## Study procedure 19 Eligible participants will be contacted via Email and welcomed into the study. Participants 20 will be asked to complete online surveys assessing primary and secondary outcomes, 21 potential mediators/moderators and socio-demographics at three time points. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] All online surveys will be administered using Qualtrics ® (Provo, Utah). If specified screening 1 criteria are not met, participants will be advised via text displayed at the end of their survey 2 and further contact will only be made where ambiguous responses require clarification. 3 Ineligible participants will also receive a link providing free and unlimited access to the 4 public version of the Balanced app. Participants will receive an Email with a unique password-protected link to their survey at 7 each assessment point. Each person who has completed their baseline survey will be 8 randomly allocated to one of two groups. Participants allocated to the intervention group will 9 be mailed a pedometer, tool sheets, login details and instructions for download and 10 installation of the "Balanced" smartphone app in the form of a participant handbook. The 11 initial Balanced app was specifically developed for scientific purposes and is described in 12 more detail elsewhere. [bib_ref] Balanced: a randomised trial examining the 8 efficacy of two self-monitoring methods..., Duncan [/bib_ref] It originally consisted of three separate categories, one for physical 13 activity (active minutes), one for inactivity (hours and minutes of sitting) and one for sleep 14 (bed and wake times and sleep quality rating). As part of the modifications to the previous 15 app, the physical activity component of the app was revised to include daily steps and 16 resistance training in addition to minutes of moderate-to-vigorous intensity physical activity; 17 and the sleep component was revised to include sleep hygiene in addition to sleep times and 18 sleep quality. The sitting behaviour category was removed for use in the Synergy study, as no 19 specific strategies to reduce sitting time will be provided in this study and because the 20 objective will be to promote improvements in moderate-to-vigorous intensity physical 21 activity and sleep health. App content was modified based on participant feedback (process 22 evaluation and semi-structured interviews) as part of the Balanced study, [bib_ref] Balanced: a randomised trial examining the 8 efficacy of two self-monitoring methods..., Duncan [/bib_ref] goals, which makes feedback on progress towards goals and goal achievement more 1 personalised and meaningful for those in need to get engaged in healthy behaviour. [bib_ref] Global health agenda on non-communicable diseases: has WHO set a smart 13..., Barreto Pds [/bib_ref] 2 3 Regular app use will be supported by an Email and text message-based support system (see [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] , which is initiated as soon as a participant has gained access to the app. All 5 messaging will follow a standardised protocol that was designed under consideration of the 6 specificity, timeliness and relevance of contents (see [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] , as those are valued 7 components in mobile apps designed to change health behaviours. Following completion of 8 their 3-month assessment, participants may continue to use the app as much or as little as 9 they like, but the message-based support will no longer be provided. ## App-based prompts If enabled, a daily on-screen notification prompts participants to log data, if app has not been used to selfmonitor behaviour in >24h x Note. The message-based support system will be delivered for the first 12 weeks of the intervention only. [bib_ref] Greater bed-and wake-time variability is 18 associated with less healthy lifestyle behaviors:..., Duncan [/bib_ref] 13 The intervention is composed of app and non-app components, with non-app components 1 referring to any content of the intervention that is delivered via participant handbook, text 2 message or Email. App components consist of educational resources, self-monitoring, goal-3 setting and feedback. Participants will have continuous access to the app throughout the 4 intervention period. For the first 3 months, which is the time between baseline and the 5 primary endpoint, these components will be complemented by a messaging system providing 6 personalised feedback on progress towards goals, prompting goal review and prompting 7 practice of the target behaviours. The messaging component will cease at the 3-month 8 assessment, but participants will have continued access to the app. Following completion of 9 the study, participants will be able to continue to access and use the app for an indefinite 10 period, however will not be required to complete any further assessments as part of this 11 study. The app will be available on both Android and Apple based operating systems. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] provides an overview of intervention strategies used to operationalise the social cognitive 13 constructs in the intervention. In brief, the key constructs included relate to a person's 14 confidence (self-efficacy) in their capacity to define and follow a specific plan, the purpose of 15 which is to experience a desired result in the face of situational or environmental (socio-16 structural) factors that either impede or facilitate progress, whilst the motivation to pursue 17 results is regulated by perceptions regarding the personal benefit of the result in question its 18 importance (outcome expectations and expectancies). Educational resources 21 App resources will consist of educational information about the importance of the two 22 behaviours, basic instructions on how to change each behaviour and guidance for app use 23 (e.g., how to interpret traffic lights and progress graphs). This content will provide 24 participants with knowledge on the health benefits of each behaviour, the current national 25 based on summaries of the evidence. In addition to app content, participants will receive a 5 total of three tool sheets (enclosed in the handbook), one tool sheet including goal-setting 6 strategies,for each behaviour, one that emphases action planning (again, one for each 7 behaviour) and one tool sheet with information and instructions adapted from publicly 8 available resources for the practice of stress management techniques (i.e., progressive muscle 9 relaxation, deep breathing and mindfulness). 42-44 All tool sheets will be distributed at outset 10 along with the participant handbook, which includes a brief study summary, a personalised 11 timeline including assessment dates as well as a comprehensive troubleshooting guide 12 covering the most common problems that may occur when installing and using the app. 13 ## Intervention 14 Participants in the intervention group will receive their materials following completion of 14 their baseline assessment and waitlist controls will receive an identical package following 15 their 6-month assessment. In addition, during each month of the intervention, one tool will be 16 promoted via Email to encourage utilisation of these resources. Goal-setting tool sheets will 17 be sent in week 3, followed by the action planning tool sheet in week 6 and the stress 18 management tool sheet in week 9 for each participant. The examples given within the sheets are framed in a way that encourages participants to tailor any strategies to their own 20 situation and priorities (for example: Once I get fitter, I will finally be able to…). Individuals 21 are instructed to set goals that are personally relevant and meaningful to promote initial 22 engagement, but the goal-setting information provided will provide reference to the 23 recommended minimum of 150 minutes of moderate-intensity physical activity per week, 7 24 and a sleep duration of 7 to 9 hours per night 45 as overarching goals one should gradually 25 work towards. Weekly summary reports however, will focus on individual progress in 1 relation to the individual goals set by the participant. Each report will detail progress in the 2 form of totals and averages for both behaviours (i.e., active minutes, step count, resistance 3 training sessions, bed and wake times, sleep hygiene, sleep quality), which will help 4 participants understand how changes in the two behaviours are interrelated. Furthermore, 5 participants will receive a weekly text message containing 1 of 12 educational and 6 motivational facts relating to physical activity and sleep for better health (i.e., the 7 consequences of poor sleep health). Each fact message will also refer to the resources section 8 available in the app and encourage people to use it. 9 10 Self-monitoring 11 Participants will be asked to recall minutes of moderate to vigorous physical activity, and 12 participation in resistance training, and manually enter this into the app every day. Daily 13 steps will be objectively measured using the pedometer (Yamax SW200, Eagle Farm, QLD) 14 provided and manually entered by participants into the app. Participants will not be asked to 15 return their pedometer. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref]. ## Operationalisation of social cognitive factors and behaviour change strategies SCT constructs BCT [bib_ref] Associations of objectively assessed physical activity 17 and sedentary time with all-cause..., Schmid [/bib_ref] Components Description of intervention components Participants will be asked to recall and enter their activity and sleep behaviours. The daily log will allow entries for active minutes, daily steps, resistance training sessions, sleep and wake times, a sleep quality rating, as well a checklist of 10 sleep hygiene goals. Participants will be asked to tick off those sleep hygiene goals they implemented the previous day. ## Self-efficacy Bar charts will provide a history for daily, weekly and 3-month progress in relation to goals per behaviour (for each of the items data are logged for). ## App dashboard traffic light The activity dashboard produces a traffic light colour relating to total active minutes, while the colour of the sleep dashboard relates to total sleep duration. Goals can be adjusted at any time, which will determine the colours on the dashboard traffic light. This is dynamically updated as soon as a self-monitoring entry is made: a green light indicates a participant is meeting, exceeding or close to their goal; an orange light indicates they are progressing toward their goal although are not close; and a red light indicates they are markedly below their goal. Tool sheets A series of tool sheets delivered at weeks 3, 6, and 9 will promote goal-setting and action planning and give detailed guidance on how to set SMART goals and follow through with an action plan in the face of barriers (i.e., by being prepared). ## Weekly summary (email) This support feature will provide an overview of weekly totals and averages per behaviour (if sufficient data are available) and prompt participants to review goals, if needed. ## Prompts (sms) If participants fail to log any data on more than 4 days per week, they will receive a message prompting them to resume logging. ## Perceived behavioural capacity Information on where and when to be active/engage in in sleep promoting behaviours Instructions on how to be active and engage in sleep promoting behaviours ## App resources The resources section will provide the current national guidelines on how much physical activity per week and how much sleep (hours) per night adults need. This section also includes brief content on the when, the where, who with and how of being active and sleeping well (e.g., sleep hygiene practices). ## Weekly facts (sms) Each week, participants will receive a short text message with educational content on activity and/or sleep and health to reinforce the importance of both behaviours. ## Tool sheets Tool sheets provide more detailed information that enable a person to make positive changes to their physical activity and sleep levels and include action plan templates and examples of exercises. These materials will also include stress management techniques, such as PMR and controlled breathing. ## Outcome expectations/ expectancies Information about the behaviour in relation to health Tool sheets As part of the goal-setting tool sheet, participants will be asked to think about the reasons for wishing to improve their health behaviours and what they anticipate as personal benefits, following improved levels of activity and sleep (examples will be provided). ## App resources App log personal goals This section will include information on why activity and sleep are important and how they contribute to health and wellbeing. Participants will be asked to personalise their goals, but work towards recommended minima (150 MVPA/week; 7-9h sleep/night); goals are carried forward from previous entries unless adjusted ## Goals Goal-setting Action Planning Self-monitoring Prompt practice Time Management Teach use of prompts Time management ## App dashboard traffic light Tool sheets Participants will be encouraged to put equal effort into improving both PA and sleep. This means 2 amber lights are better than one green and one red light. Participants will receive goal-setting strategies and example action plans for guidance (per behaviour) as part of the tool sheets described above. One of 3 tools will be promoted specifically via Email at week 3, 6 and 9, respectively. ## Reminders Participants are advised to set a daily bedtime reminder (optional) on their phone, which is intended to prompt a person's bedtime routine and will promote regular bed times. App resources Environmental restructuring as part of good SH will be highlighted in the resource section and include details on how to manage the bedroom environment. Also includes information on activity & sleep in the social context and seeking support from those in the same household (housemates, partner, family members). ## Sociostructural factors (social support & environment) Use of prompts Environmental restructuring Barrier identification Plan social support Self-monitoring of sleep in the app will also be manually entered by participants. The sleep 1 log consists of: bedtime (time of going to sleep), wake time (time of waking) and sleep 2 quality (rating scale from 0 to 5 where 5 indicates high sleep quality). As an additional 3 feature, this section of the app allows participants to log which sleep hygiene behaviours they 4 practiced the previous day [fig_ref] Figure 2: Sleep review only -http [/fig_ref]. These include consumption of caffeine, alcohol, 5 nicotine, excessive intake of fluids or heavy meals before bedtime, regulation of the impact 6 of light, noise and temperature in the bedroom, use of light-emitting devices, regular exercise, 7 maintenance of consistent sleep and wake times, having and following a bedtime routine, 8 creating comfort (e.g., proper pyjamas and bedding) and managing stress. 22 Participants can 9 self-monitor these behaviours at any time of the day and update this information as many 10 times per day as they prefer. The current study uses a manual data entry method based on 11 self-monitoring. This method was selected for use in the current trial due to financial 12 restrictions and because the Balanced study did not observe any between-group differences 13 (i.e., manual entry vs. device-entered method (via Fitbit) in physical activity or sleep 14 outcomes, or in time to non-usage attrition. [bib_ref] Balanced: a randomised trial examining the 8 efficacy of two self-monitoring methods..., Duncan [/bib_ref] Self-regulation 17 App feedback on behaviour will be provided using graphical displays of logged behaviour in 18 relation to the goals set by the participant [fig_ref] Figure 3: Screenshots of app screens for self-monitoring and feedback relative to goals 209x148mm [/fig_ref]. Two types of graphical feedback are 19 provided. There will be separate graphs for moderate-to-vigorous intensity physical activity, 20 steps, resistance training, sleep duration, sleep quality, sleep timing and sleep hygiene. This 21 information will provide a breakdown in the form of daily, 1-week and 3-month bar charts. 22 The second graphical feedback to participants is via the dashboard which changes to one of 23 three colours -green, orange and red in a traffic light system -to provide immediate feedback 24 on participants' behaviour to in relation their goals on a daily basis [fig_ref] Figure 3: Screenshots of app screens for self-monitoring and feedback relative to goals 209x148mm [/fig_ref]. The 25 the use of consistent criteria across behaviours. This differs to the traffic light system 2 originally used in Balanced, since process data from that study alluded to participants 3 preferring to see this feedback based on goals rather than guidelines for each behaviour. As part of the goal review strategies, participants will be encouraged to evaluate their 6 achievements in relation to goals and adjust their goals whenever needed. This will be 7 facilitated by a personalised weekly summary of the previous week, delivered via Email, so 8 that any reviews and adjustments of goals align with the most recent progress and foster self-9 efficacy. If a participant has logged data on less than 4 days per week (per behaviour), a text 10 message will be sent to prompt practice. Likewise, once per week, if a participant only logs 11 data for one behaviour, but not the other, a prompt will encourage him or her to engage in 12 both behaviours equally. [bib_ref] Sleep and society: an epidemiological perspective, Bixler [/bib_ref] 14 ## Waitlist control group 15 Upon enrolment and allocation, the waitlist control group will not receive any intervention 16 materials and only be required to complete their baseline, 3-month and 6-month assessments. 17 After the 6-month assessment is completed, participants in this group will receive full access 18 to the intervention. 19 20 ## Randomisation 21 Participants will be randomly allocated to 2 groups (intervention or control) after having 22 completed their baseline assessment. Opaque sealed envelopes (n = 80 per group) will be 23 prepared by BM using permuted block randomisation with block sizes of 4 and 8, following 24 the procedures suggested by Doig et al. [bib_ref] Randomization and allocation concealment: a practical guide for 34 researchers, Doig [/bib_ref] Once a participant has completed their baseline 25 will open the envelope that is next in sequence and inform the project leader about the 2 allocation outcome. Participants will be informed by the project leader and be sent a package 3 containing study materials (i.e., handbook and pedometer), if they have been allocated to the 4 intervention group (participants in the waitlist control group will receive their study materials 5 after completing their 6-month assessment). The only exception for contravention with the 6 allocation sequence will be made if family members or couples living in the same household 7 enrol in the study, which would pose a high risk of contamination, especially between groups. 8 For this reason, all individuals who are identified as members of the same household will be 9 allocated to the same group. Neither the trial participants, nor the project lead (BM) will be 10 blinded to group assignment. 11 12 ## Outcome measures 13 All measures will be assessed via online survey at baseline, 3 months, and 6 months, except 14 for socio-demographics which will only be collected at baseline. The 3-month survey will 15 further include process evaluation items that measure system usability and participant 16 satisfaction (intervention group only). The two primary outcomes will be total minutes of 17 moderate-to-vigorous physical activity and sleep quality. To increase adherence to scheduled 18 assessments, participants who complete their survey will be entered into a draw for 1 of 5 $50 19 shopping vouchers. This information will not be provided prior to enrolment and is not 20 intended to function as an incentive for individuals to sign up to participate, but merely to 21 promote adherence to assessment requirements. [fig_ref] Table 3: Overview of outcome measures and assessment time points [/fig_ref] provides a summary of outcome 22 measures and assessment time points. All online surveys will be pilot-tested and locked prior 23 to study commencement to prevent any changes from being made once the study is 24 underway. All survey forms will be hosted on Qualtrics ® . 25 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref]. week. This includes the total time spent in recreational walking and transport, moderate-6 intensity physical activity (e.g., swimming, golfing), aerobic activity (e.g., cycling, jogging) 7 and vigorous gardening or yard work. Total minutes of moderate-and vigorous-intensity 8 physical activity will be created by summing minutes of walking, moderate and vigorous 9 (weighted by 2) intensity physical activity. Although objective assessment methods may be 10 used to measure physical activity, it was not deemed feasible in the current study due to 11 financial and pragmatic issues. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] global measure of subjective sleep quality will be used in this study to observe the perceived 1 restorative effect of sleep, which is difficult to measure using objective methods. 57 2 3 ## Overview of outcome measures and assessment time points ## Secondary outcomes 4 Health-related quality of life 5 Poor sleep quality and inadequate sleep duration are independently associated with low 6 health-related quality of life. 58 The RAND-12 is a valid and reliable instrument 59 that is 7 widely used to assesses multiple concepts of health such as physical functioning, role 8 limitations due to physical and emotional problems, social functioning, emotional wellbeing, 9 energy/fatigue, pain as well as general perceptions of health. In addition to the RAND-12 10 scale, 3 additional items that make up the energy/fatigue subscale in the 36-item version of 11 the RAND will be asked (e.g., "How much of the time during the past 4 weeks did you feel 12 tired?"), so that this domain can be evaluated separately. This will allow improvements in 13 energy and fatigue during the course of the intervention to be assessed. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Since the AAQ does not capture resistance training and because the Synergy Study will 1 promote regular resistance training, the number and duration of resistance training sessions 2 per week will be assessed using two items adapted from previous studies that assessed 3 resistance training. 61 One item will ask participants: "In the last week, on how many days 4 have you participated in muscle strengthening activities (including weight/resistance 5 training)?" and "What do you estimate was the total time (in hours/minutes) that you spent 6 doing muscle strengthening activities (incl. weight/resistance training) in the last week?" The 7 original items were adapted by changing the recall period from the previous month to the last 8 week to align with the recall period used in the AAQ. 9 10 Sitting time 11 The Workforce Sitting Questionnaire (WFSQ) will provide a self-report measure of total 12 domain-specific sitting time (over the last week), on workdays and non-workdays. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Internet self-efficacy 2 Participants' confidence in using the smartphone app will be assessed using an adaptation of 3 the Internet Self-Efficacy Scale to capture participants' overall understanding of app 4 software, confidence in gathering information using the app and learning to use the app, as 5 well as the ability to troubleshoot and resolve app problems. [bib_ref] Coursaris CK, van Osch W. A Cognitive-Affective Model of Perceived User Satisfaction..., Eastin [/bib_ref] Participants will rate their 6 agreement using a total of eight statements (e.g., "I feel confident explaining why a task will 7 not run on the smartphone/tablet") on a 7-point scale from strongly disagree to strongly 8 agree. 9 10 Perceived user satisfaction 11 The Cognitive-Affective Model of Perceived User Satisfaction (CAMPUS) will be used to 12 ask participants about thoughts and feelings associated with using the mobile app (Balanced). 13 Using a 7-point scale ranging from strongly disagree to strongly agree, a total of 23 items 14 enquire about participant opinion on the effects and aesthetics as part of the app design items), its effectiveness and efficiency (5 items) and the level of satisfaction experienced 16 when using the app (3 items) with the following anchors: frustrated -contented, unhappy -17 gratified and sad -joyful. Items will be adapted to refer specifically to the Balanced app, for 18 example "I would consider my experience with using the Balanced app as innovative". This 19 instrument has demonstrated adequate levels of reliability and validity. 68 20 21 ## App usage 22 Overall interaction with the app will be measured continuously throughout the study period 23 by the app database, which records the time and date a self-monitoring entry was made and 24 the actual value or response entered into the app. Analysis of usage patterns will include the 25 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] App usability will be assessed using the 10-item System Usability Scale, 71 a valid and 6 reliable tool that assesses participant satisfaction relating to the utility of the websites on a 5-7 point scale (items will be reworded for smartphone app usability). Higher total scores (range 8 1-100) relate to better usability. Example items include "I would imagine that most people 9 would learn to use this system very quickly." or "I needed the support of a technical person to 10 be able to use this system.". 11 12 ## Utility, advice acceptability and relevance of the app 13 A participant sub-sample (10%) will be determined by random selection for semi-structured 14 telephone interviews, which will take place once all participants have completed their 6-15 month assessments. These interviews will contribute valuable information for process 16 evaluation and include general personal feedback, desirable improvements and preferences 17 relating to future use. As part of these interviews, participants will be asked about their 18 perception of the app's usefulness to improve changes in self-efficacy levels (confidence) 19 toward physical activity and sleep health, coping with potential impediments (barriers) to 20 being more active and sleeping better, maintaining new routines/action plans and keeping it a 21 priority to be more active and sleeping better. Finally, advice acceptability and relevance in 22 terms of the content will be examined based on a previously used questionnaire. 72 23 24 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Testing social cognitive factors as potential mediators of intervention efficacy may provide 2 insights into some of the underlying mechanisms of behaviour change, as observed in 3 previous health behaviour interventions. [bib_ref] Testing mediator variables in a physical 25 activity intervention for women with..., Plotnikoff [/bib_ref] Constructs from Social Cognitive Theory 29 will be 4 assessed using partially modified items from previously developed scales, with distinct items 5 per behaviour relating to the person's projections towards the occurrence of each behaviour 6 over the next three months. The constructs of interest include self-efficacy, perceived 7 behavioural capacity, outcome expectations and expectancies, goals, action planning and 8 socio-structural factors including social support and the environment. Items are described in 9 more detail below and [fig_ref] Table 4: Social cognitive factors related to physical activity and sleep hygiene behaviours [/fig_ref] summarises the number of items per behaviour per construct 10 and lists response options for each item. 11 ## Mediators and moderators 25 ## 12 Physical Activity Items 13 For physical activity, a total of 34 items will be used to assess the social cognitive factors and 14 a sum score will be calculated for each construct. Prior to asking these questions, participants 15 will be advised that in the context of these questions "regular physical activity is defined as 16 doing at least 150 minutes of moderate intensity physical activity each week. Moderate 17 intensity can be described as any type of aerobic activity performed at a level where a person 18 begins to lightly sweat, but can still carry on a conversation. This may feel different from one 19 person to another." 20 21 Self-efficacy 22 Self-efficacy levels in the context of barriers will be assessed using a modified version of 23 validated measures 74 consisting of 10 items. Response choices for these items range from 24 "not at all confident" (1) to "extremely confident" (5) and items share the same stem ("I am 25 Participants will be asked how confident they are about having the capacity to engage in 9 specific amounts and intensities of physical activity, using 3 statements 75 and response 10 options from "never" (1) to "always" [bib_ref] Sleep duration and risk of type 2 diabetes: a meta-analysis of 32..., Shan [/bib_ref]. An example statement is: "I can run or jog for 10 11 minutes without stopping." 12 [bib_ref] Sleep and society: an epidemiological perspective, Bixler [/bib_ref] Outcome expectations and expectancies 14 Using 5 items per construct, a total of 10 items will assess participants' expectations and 15 expectancies pertaining to perceived personal gains (outcome expectations) from engaging in 16 regular physical activity, followed by the level of importance associated with these gains 17 (outcome expectancies). On a 5-point Likert scale ("strongly disagree" to "strongly agree"), 18 participants will be asked first to indicate their level of agreement with 1 of 5 statements 19 (adapted from relating to perceived benefits of regular engagement in 20 physical activity (e.g., "Being physically active can reduce my risk for some illnesses and 21 diseases (e.g., heart disease, diabetes, some cancers, etc.).") and then rate the value this 22 would have for themselves (e.g., "How important is [e.g., reducing your risk for illness and 23 disease?]") on a 4-point Likert-type scale ("not at all important" to "extremely important"). 24 The role a person's social network plays in influencing physical activity participation will be 5 assessed by asking participants about their level of agreement (on a 7-point scale from 6 "strongly disagree" to "strongly agree") with 2 items that were previously modified for use in 7 the context of a physical activity intervention 77 : "People in my social network are likely to 8 help me participate in regular physical activity." and "I feel that someone in my social 9 network will provide me with the support I need to get regular physical activity.". 10 11 Environment 12 The impact a person's built and natural environment has on physical activity engagement will 13 be measured using 3 items from the IPAQ environmental module [bib_ref] Sleep health: can we define it? Does it matter?, Buysse [/bib_ref] To further assess the motivational mechanisms that drive progress towards goal attainment , 1 participants will be asked to indicate the extent to which they intend to be active on a regular 2 basis using 2 adapted items: "Do you intend to do regular physical activity over the next 3 3 months?" 76 and "How motivated are you to do regular physical activity over the next months?" 79 that are answered using 7-point Likert-type response choices ranging from "No, 5 not really" (1) to "strongly intend" (7) and "Not at all motivated" (1) to "Extremely 6 motivated" (7), respectively. For both items, higher scores indicate greater strength of goals 7 and the 2 scores will be summed to obtain a total score for goals. 8 9 Planning 10 Plans concerning "when", "where", "how" and "what kind" of physical activity participants 11 will engage in will be assessed using a previously modified scale 80 that consists of respectively worded items, where higher scores are interpreted as more detailed planning 13 ("no plans" (1) -"detailed plans" (7)). 14 15 ## Sleep hygiene items 16 To assess the same constructs as above in the context of sleep hygiene practice, a total of 72 17 items were developed using partially modified scales that were previously used to assess right before bedtime or in bed. To avoid overburdening participants, each construct will be 1 assessed using a single item per sleep hygiene behaviour. Thus, each social cognitive 2 construct will have nine items. Each construct will be scored as the sum of the nine sleep 3 hygiene items, with a higher sum score indicating improvement. The environment construct 4 however, will not be included for sleep hygiene behaviours, as this is already captured as part 5 of the perceived neighbourhood disorder questionnaire described below. 6 7 Self-efficacy 8 Items assessing self-efficacy relating to sleep hygiene will be answered on a 5-point Likert-9 type scale ("not at all confident" to "extremely confident) using the commonly used stem "I To assess social support as a socio-structural factor that may or may not have a facilitating 10 effect on sleep hygiene practice, the commonly used stem "Most people who are important to 11 me would encourage me to [e.g., reduce my stress levels.]" 79 82 will be used with response 12 choices ranging from "strongly disagree" (1) to "strongly agree". 13 ## 14 ## Goals 15 The extent to which people "intend to […]" practice sleep hygiene behaviours will be 16 measured using a 7-point Likert-type scale (from "no, not really" to "strongly intend") with 17 higher scores indicating stronger goals. This item was used previously in a sleep hygiene 18 context. Planning 21 To test participants' plans with regards to practicing good sleep hygiene, each of the nine 22 items assessing this construct will ask if a person has planned "where, when and how" to 23 avoid caffeine, avoid nicotine, avoid alcohol, exercise regularly, reduce their stress levels, 24 minimise the impact of noise and nuisance in their bedroom, keep their sleep and wake times 25 (1) no, not really (7) strongly intend and Action planning 9 (1) no detailed plans (7) detailed plans Note. Each item per construct will refer to one of nine different sleep hygiene behaviours 1 Automaticity 2 Habits relating to lifestyle behaviours are non-conscious processes, which can act as 3 determinants of behaviour and may even regulate behaviour independently of changes in 4 conscious processes such as implementation intentions (goals). The role that behavioural 5 automaticity plays in the context of physical activity and sleep behaviours, respectively, will 6 be taken into account using 1 item from the Automaticity Index per sleep hygiene behaviour 7 (9 items), [bib_ref] Towards parsimony in habit measurement: testing 11 the convergent and predictive validity..., Gardner [/bib_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] behaviours. [bib_ref] Neighborhood disorder, psychophysiological distress, and 30 health, Hill [/bib_ref] Based on an existing scale of neighbourhood disorder, which demonstrated 5 good levels of construct validity and internal consistency/reliability, 90 4 items will assess 6 each of the following characteristics of neighbourhood disorder: physical disorder and 7 physical order, social disorder and social order. These are assessed using the following 8 items: (1) "My neighbourhood is noisy", (2) "My neighbourhood is clean", (3), "There is a 9 lot of crime in my neighbourhood" and (4) "My neighbourhood is safe" These items will be 10 answered on a 5-point scale from strongly disagree (1) to strongly agree (5) and the average 11 responses across the four items will be calculated. 12 ## 13 ## Sample characteristics 14 A range of demographic and socioeconomic factors such as age, gender, height and weight, 15 education, income, marital status, occupation, working hours, etc. will be assessed. 16 Participants will be asked to also indicate (allowing multiple selection) whether they have 17 been told by a doctor that they have any of the following chronic diseases: arthritis, asthma, 18 cancer, cerebrovascular disease (stroke), chronic obstructive pulmonary disease 19 (emphysema), coronary heart disease (heart attack, angina), type-1 diabetes, type-2 diabetes, 20 high blood pressure, kidney disease, mental illness (depression, anxiety, etc.), osteoporosis, 21 irritable bowel syndrome, high cholesterol, or any other disease (to be specified by the 22 participant). In addition, participants will be assessed for morningness or eveningness type, as eveningness types are thought to be more prone to engage in activities that cause social 24 jetlag, due to misalignments between times of sleep and times of social activity. [bib_ref] Social jetlag: misalignment of biological and 39 social time, Wittmann [/bib_ref] Meta-analyses of physical activity interventions typically report small to moderate increases 3 in physical activity (Cohen's d = 0.14 -0.68). Moreover, poor sleep health, specifically 4 the duration or quality of sleep, has small to moderate magnitude associations with lower 5 physical activity levels. 95 Meta-analyses of non-pharmacological sleep interventions report 6 small to medium effect sizes for changes in sleep quality (Hedge's g = 0.35 and Cohen's d = 7 0.41) in clinical populations, and medium to large effect sizes (d = 0.74) in studies using 8 exercise to improve sleep. 98 Therefore, based on these observations and feasibility data from 9 a previous study, [bib_ref] Balanced: a randomised trial examining the 8 efficacy of two self-monitoring methods..., Duncan [/bib_ref] it was assumed that a 3-month combined physical activity and sleep 10 intervention that specifically targets both behaviours and leverages the bi-directional 11 relationship between behaviours is likely to produce moderate increases in physical activity 12 Meta-analyses of physical activity and sleep interventions report average drop-out rates of 23 20%, however, the majority of web-based trials report drop-out rates that are higher than 24 that. [bib_ref] Meta-analysis of internet-delivered 42 interventions to increase physical activity levels, Davies [/bib_ref] As there is insufficient information available on attrition in m-health interventions, the 25 sample size for this study will be inflated to account for a 25% drop-out. Therefore, the total 1 sample size is 80 participants per group or 160 in total. A sample of this size will also be 2 adequately powered to detect mediated effect sizes of small (β = 0.14) magnitude.The 3 participant recruitment phase will conclude once 160 complete baseline responses have been 4 obtained. 5 6 Analyses 7 Analyses will apply the intention-to-treat principle. Analysis of primary outcomes will be 8 blinded to group allocation and overseen by an independent statistician. The primary aim of 9 this study will be to examine differences in physical activity and sleep quality between the 10 intervention group and the control group at the 3-month primary time point. Between-group 11 differences in physical activity (AAQ minutes) and sleep quality (PSQI) will be estimated 12 using Generalised Linear Mixed Models (GLMM) adjusting for baseline measures of the 13 outcome, including all available data in the analysis. The model will include fixed effects for 14 group, time and their interaction. A random intercept will be used to account for repeated 15 measures on individuals. Separate GLMM will be used to examine changes in physical 16 activity and sleep. 17 [bib_ref] Sleep and exercise: a reciprocal issue, Chennaoui [/bib_ref] Sensitivity analyses using Pattern Mixture Modelling will be conducted to examine the 19 impact of missing data on outcomes. Where the GLMM assumes data are missing at random, 20 Pattern Mixture Modelling is robust to the assumed pattern of missing data. Group 21 differences in secondary outcome measures will be estimated using the same linear mixed 22 modelling approach, setting an alpha of 0.025 for each outcome. Potential mediators and 23 moderators of intervention efficacy will be examined using established approaches. [bib_ref] A general approach to causal mediation analysis, Imai [/bib_ref] Any type of adverse events reported by study participants that occurred in relation to their 5 participation in the study will be recorded and reported to the HREC. This may include 6 events reported by participants, including musculoskeletal injuries associated with the uptake 7 or increase in physical activity or emotional distress due to any survey items of sensitive 8 nature. Great care will be taken to avoid and prevent adverse events and the research team 9 will provide every possible assistance to remediate those events, should they occur. The 10 participant information statement interested individuals will have access to prior to 11 consenting to participate details any potential risks of discomfort associated with 12 participation in the study and provides contact details and information of national support 13 services (e.g., Black Dog Institute, Lifeline, etc.). 14 15 Survey data will be exported directly from Qualtrics ® as a text file and imported in electronic 16 form for scoring and analysis using statistics software. A detailed database will track 17 participants' progress through the trial including the scheduling of assessments and reminders 18 to complete assessments. Intervention usage will be monitored throughout the trial by and MJD by way of the password-protected app database. Given the purpose of the trial, the 20 data to be collected as well as the nature of the intervention, no Data Monitoring Committee 21 will be established. Detailed strategies, including Email/text message reminders will be used 22 to remind participants about upcoming assessments. All Newcastle-based members of the 23 research team (BM, MJD, ATR, RCP) and other associated personnel will have access to the 24 information in both identified and re-identifiable forms. Should statistical analysis advice be 25 Results from the outcome measures will not be presented in a way that adversely affects the 11 confidentiality of participants. The description of participants will not allow identification of 12 individual participants, and individual results and individual names will not be revealed. 13 Final reports and publications will only consist of aggregated results. At the completion of 14 the study, participants will receive a plain English summary of study results. Scientific 15 reports of the main outcomes, secondary outcomes and process evaluation will be submitted 16 to peer-reviewed journals. Results will also be incorporated into student theses and presented 17 at national and international conferences. 18 ## 19 # Discussion 20 It is advised that adults accumulate a weekly minimum of 150 minutes of moderate intensity 21 physical activity, combined with muscle strengthening activities on two days per week, 7 and 22 also achieve 7-9 hours of good quality sleep each night. 101 Engaging in the recommended 23 levels of physical activity and maintaining good sleep health contributes to overall health and 24 wellbeing through risk reductions associated with chronic diseases such as heart disease and 25 behaviour interventions suggested that those targeting related behaviours (e.g., diet and 17 physical activity) produced greater behaviour change than those targeting unrelated 18 behaviours (e.g., smoking and physical activity), 111 and that specific intervention techniques 19 are necessary for each behaviour. [bib_ref] Do single and multiple behavior change 17 interventions contain different behavior change..., Mc Sharry [/bib_ref] Physical activity and sleep are suggested to have a bi-20 directional relationship, [bib_ref] The bidirectional relationship between exercise and sleep: Implications for 32 exercise adherence..., Kline [/bib_ref] yet no previous RCTs have combined physical activity and sleep in 21 one intervention and therefore have not utilised the synergistic relationship between physical 22 activity and sleep. The Synergy Study addresses this by targeting both physical activity and 23 sleep, simultaneously, using specific intervention techniques to enhance participants' self-24 regulatory skills in relation to the two health behaviours and thus, leverages the potential for 25 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] synergistic improvements in both behaviours. An advantage of this study lies in its mode of 1 delivery, which involves mobile technology and therefore blends into day-to-day life. A key 2 intervention strategy is the use of goal-setting and feedback to promote behaviour change. It 3 seeks to achieve this through the promotion of dynamic goals and action plans, the 4 implementation of a personalised support system further addresses potential barriers (i.e., low 5 levels of self-efficacy) that can increase the gap between participant intentions (goals and 6 plans) and behavioural outcomes, 112 and contribute to long-term behaviour maintenance. This 7 includes knowledge on how to set attainable goals and having strategies in place that 8 facilitate the occurrence of healthy behaviours, despite challenging situations or unfavourable 9 environmental factors. The structured promotion of goal-setting strategies, combined with 10 action plans that define in detail how an individual will implement the intended behaviour, is 11 known to be effective in changing health behaviours. Additional strengths of this study include its randomised waitlist controlled study design, 14 which will allow making inferences about the causal links between the intervention and 15 changes in behaviour. The use of remote delivery through a m-health format makes it 16 possible to recruit nationwide and has the potential to be scaled up further including an 17 international version of the programme. Delivering the Synergy study in multiple countries 18 however, would require further refinement of the contents and adaptation to cross-cultural 19 factors as well as geographical differences. While it is the first aim of this study to test the 20 intervention's efficacy to produce changes in two primary outcomes, the pre-specified 21 secondary outcomes (mental health, quality of life) will give insight into changes in 22 parameters of health and wellbeing that may be very meaningful to the participant. And 23 finally, this study will generate knowledge on social cognitive determinants of behaviour 24 change relating to sleep health and explore how these factors differ between physical activity 25 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] and sleep. This will enhance the understanding of underlying mechanisms associated with 1 successful behaviour change in both behaviours and also further the application of social 2 cognitive theories in the multi health behaviour context. The limitations of this study include 3 the study duration, which, although at 6 months is longer than many studies, [bib_ref] Meta-analysis of internet-delivered 42 interventions to increase physical activity levels, Davies [/bib_ref] does not 4 provide insight into longer term changes and behaviour maintenance; and the lack of a 5 comparator condition receiving only the sleep or the physical activity programme to 6 determine the magnitude each intervention component has on its own. It is beyond the scope 7 of this study to test long-term efficacy exceeding 6 months, but future trials may be 8 encouraged to do this, provided the Synergy Study proves efficacious in the short term with 9 indications of effect retention at the 6-month time point. 10 ## 11 # Conclusion 12 This study protocol provides the rationale and methods associated with the implementation 13 and evaluation of the Synergy Study, a theory-based m-health intervention including 14 personalised support, with the aim to improve physical activity and sleep health in adults. To the authors' knowledge, this study will be the first to simultaneously target 16 changes in these two behaviours, using a sophisticated combination of technologies and 17 evidence-based strategies and test the efficacy of this approach in a randomised controlled 18 trial. Findings from this trial will provide valuable knowledge pertaining to the design of m-19 health interventions that combine behaviours in a format with wide reach. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] The trial is sponsored by the University of Newcastle and will be coordinated independently 2 of the study sponsor and funder by the Priority Research Centre for Physical Activity and 3 Nutrition, University of Newcastle, Australia and managed by project lead BM and overseen 4 by chief investigator MJD. The study funder and sponsor will have no role in the conducting 5 or evaluation of the trial, nor did the study funder and sponsor have any authority over the 6 study design, collection, management, analysis and interpretation of data, the preparation of 7 manuscripts or the submission of reports. 8 9 ## Declaration of interests 10 The authors and principal investigators of this study protocol declare no conflict of interest. 11 12 ## Roles and responsibilities 13 Any amendments to the study protocol will be submitted to the Human Research Ethics 14 Committee (HREC) and updated on the trial register (ANZCTR) once full ethical approval 15 has been obtained. All authors meet ICMJE criteria for authorship in that they have 16 contributed substantially to the conceptual design; or the processes of data collection, 17 analysis or interpretation; the drafts and revisions of the study protocol and manuscript; 18 granted approval of the final version of the study protocol; and acknowledged their 19 accountability with regards to the integrity and accuracy of this study protocol. In detail, 20 the first author of this protocol (BM) will be responsible for administrative and managerial 21 procedures related to all phases of the trial, which will be supervised by MJD and RCP. ATR [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref]. Overview and content of message-based support service 5 . Operationalisation of social cognitive factors and behaviour change strategies 6 [fig_ref] Table 3: Overview of outcome measures and assessment time points [/fig_ref]. Overview of outcome measures and assessment time points 7 [fig_ref] Table 4: Social cognitive factors related to physical activity and sleep hygiene behaviours [/fig_ref]. Social cognitive factors related to physical activity and sleep hygiene behaviours [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] ## Scientific title A randomised controlled trial using a theory-based m-health intervention to improve physical activity and sleep health in adults: The Synergy Study protocol. ## Countries of recruitment australia ## Health condition(s) or problem(s) studied Physical inactivity and poor sleep health will be targeted to promote the prevention of chronic diseases such as type-2 diabetes, heart disease and obesity through greater levels of physical activity and better sleep health (characterised by optimal sleep duration, good sleep quality and consistent bed and wake times). If the study is conducted in healthy human volunteers belonging to the target population ## Intervention(s) Intervention Group: The intervention group will receive a combined physical activity and sleep intervention using a mobile app and personalised support for 3 months Intervention Description: The mobile app will operationalise goal-setting, self-monitoring and feedback strategies in relation to physical activity and sleep health. The messagebased support service (Email and text messaging) will provide feedback on progress towards goals, prompts to practice the two behaviours and review/adjust goals, daily reminders (optional) and tool sheets on goal-setting, action planning and stress management. Participants will be asked to self-monitor their daily physical activity and sleep behaviours and set personal goals for both behaviours within the app. We will advise participants to log both behaviours on a daily basis, whereas goals can be reset at any time (if need be) or otherwise will remain unchanged. A summary report, which is sent to participants once weekly will assist with the process of goal review. Goal achievement is considered successful, if participants are within 20% of their goal. For example, if a participant aimed for 30 min of activity, their feedback dashboard (traffic light) will display a green light and their goal is reached, if the participant logged at least 24 min of activity or more for that day. Multiple entries per day are possible. A pedometer will be shipped to participants to facilitate the logging of a daily step count. Participants will be asked to set relevant and achievable goals (guidance provided); however, we will encourage individuals to gradually work towards the recommended minimum of physical activity (PA) and sleep as per national guidelines, which is 150 min/week (plus 2 days of resistance training) for PA and 7-9 hours of sleep per night. The app and all other components are informed by social cognitive theories and follow behaviour change taxonomies to ensure consistency with implementation standards and reporting. Participants will be given continuous access to the app. Waitlist Control Group: Following the 6-month follow-up assessment, thee waitlist control group will receive full access to the intervention [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] o planning frequent travel (once a month or more often) to a destination with a shift in o time zone by more than three hours during the intervention period; o currently using a self-monitoring system or device to track or log physical activity or o sleep (this includes non-device assisted applications); and o not having access to an internet-enabled iOS (Apple) or Android smartphone or tablet. ## Key inclusion and exclusion criteria ## Study type Study type consists of: o Type of study: interventional o Study design: 2-arm RCT with a waitlist control group Method of allocation: randomized Masking: unmasked Assignment: intervention group vs. waitlist control group Purpose: to determine causal effect of the intervention o Phase: n/a o Allocation concealment and sequence: Opaque envelopes (k = 80 per group) using permuted block randomization with block sizes of 4 and 8. A researcher not associated with the study who is responsible for group allocation will open the envelope that is next in sequence and inform the project leader about the allocation outcome. [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Role of study sponsor and funders, if any, in study design; collection, management, analysis, and interpretation of data; writing of the report; and the decision to submit the report for publication, including whether they will have ultimate authority over any of these activities 32 5d ## Date of first enrolment Composition, roles, and responsibilities of the coordinating centre, steering committee, endpoint adjudication committee, data management team, and other individuals or groups overseeing the trial, if applicable (see Item 21a for data monitoring committee) 32 Trial design 8 Description of trial design including type of trial (eg, parallel group, crossover, factorial, single group), allocation ratio, and framework (eg, superiority, equivalence, noninferiority, exploratory) 6 # Introduction ## Methods: participants, interventions, and outcomes Study setting 9 Description of study settings (eg, community clinic, academic hospital) and list of countries where data will be collected. Reference to where list of study sites can be obtained 6 Eligibility criteria 10 Inclusion and exclusion criteria for participants. If applicable, eligibility criteria for study centres and individuals who will perform the interventions (eg, surgeons, psychotherapists) 6f Interventions 11a Interventions for each group with sufficient detail to allow replication, including how and when they will be administered 9ff 11b Criteria for discontinuing or modifying allocated interventions for a given trial participant (eg, drug dose change in response to harms, participant request, or improving/worsening disease) 17 11c Strategies to improve adherence to intervention protocols, and any procedures for monitoring adherence (eg, drug tablet return, laboratory tests) Relevant concomitant care and interventions that are permitted or prohibited during the trial n/a Outcomes 12 Primary, secondary, and other outcomes, including the specific measurement variable (eg, systolic blood pressure), analysis metric (eg, change from baseline, final value, time to event), method of aggregation (eg, median, proportion), and time point for each outcome. Explanation of the clinical relevance of chosen efficacy and harm outcomes is strongly recommended 19ff Participant timeline 13 Time schedule of enrolment, interventions (including any run-ins and washouts), assessments, and visits for participants. A schematic diagram is highly recommended (see 8 [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Strategies for achieving adequate participant enrolment to reach target sample size 6 ## Methods: assignment of interventions (for controlled trials) Allocation: Sequence generation 16a Method of generating the allocation sequence (eg, computer-generated random numbers), and list of any factors for stratification. To reduce predictability of a random sequence, details of any planned restriction (eg, blocking) should be provided in a separate document that is unavailable to those who enrol participants or assign interventions 17 Allocation concealment mechanism 16b Mechanism of implementing the allocation sequence (eg, central telephone; sequentially numbered, opaque, sealed envelopes), describing any steps to conceal the sequence until interventions are assigned 17 Implementation 16c Who will generate the allocation sequence, who will enrol participants, and who will assign participants to interventions 17 Blinding (masking) 17a Who will be blinded after assignment to interventions (eg, trial participants, care providers, outcome assessors, data analysts), and how 17 17b If blinded, circumstances under which unblinding is permissible, and procedure for revealing a participant's allocated intervention during the trial n/a ## Methods: data collection, management, and analysis Data collection methods 18a Plans for assessment and collection of outcome, baseline, and other trial data, including any related processes to promote data quality (eg, duplicate measurements, training of assessors) and a description of study instruments (eg, questionnaires, laboratory tests) along with their reliability and validity, if known. Reference to where data collection forms can be found, if not in the protocol 18ff 18b Plans to promote participant retention and complete follow-up, including list of any outcome data to be collected for participants who discontinue or deviate from intervention protocols [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Plans for data entry, coding, security, and storage, including any related processes to promote data quality (eg, double data entry; range checks for data values). Reference to where details of data management procedures can be found, if not in the protocol 30 Statistical methods 20a Statistical methods for analysing primary and secondary outcomes. Reference to where other details of the statistical analysis plan can be found, if not in the protocol 30 20b Methods for any additional analyses (eg, subgroup and adjusted analyses) 30 20c Definition of analysis population relating to protocol non-adherence (eg, as randomised analysis), and any statistical methods to handle missing data (eg, multiple imputation) 30 # Methods: monitoring Data monitoring 21a Composition of data monitoring committee (DMC); summary of its role and reporting structure; statement of whether it is independent from the sponsor and competing interests; and reference to where further details about its charter can be found, if not in the protocol. Alternatively, an explanation of why a DMC is not needed 31 21b Description of any interim analyses and stopping guidelines, including who will have access to these interim results and make the final decision to terminate the trial n/a ## Harms 22 Plans for collecting, assessing, reporting, and managing solicited and spontaneously reported adverse events and other unintended effects of trial interventions or trial conduct 33 Auditing 23 Frequency and procedures for auditing trial conduct, if any, and whether the process will be independent from investigators and the sponsor n/a [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] Consent or assent 26a Who will obtain informed consent or assent from potential trial participants or authorised surrogates, and how (see 6 26b Additional consent provisions for collection and use of participant data and biological specimens in ancillary studies, if applicable n/a ## Ethics and dissemination ## Confidentiality 27 How personal information about potential and enrolled participants will be collected, shared, and maintained in order to protect confidentiality before, during, and after the trial 31 ## Declaration of interests 28 Financial and other competing interests for principal investigators for the overall trial and each study site 32 Access to data 29 Statement of who will have access to the final trial dataset, and disclosure of contractual agreements that limit such access for investigators [fig_ref] Table 1: Overview and content of message-based support service [/fig_ref] 48 [fig] •: the high prevalence of people who report either being insufficiently active or meeting 2 indicators of poor sleep health, there is a need for broad reaching interventions. Because 3 smartphone ownership is growing steadily, with approximately 80% of the population 4 owning a device, 33 intervention delivery entailing this medium is likely to be accessible, 5 affordable and conveniently integrated into daily life. currently pregnant or having given birth in the past 12 months; 23 having a condition that would make it unsafe or limits their ability to increase activity 24 levels or change sleep behaviours; [/fig] [fig] 13: 13-item Sleep Hygiene Index (SHI) developed by Mastin et al. 76 Higher global scores 14 indicate poorer sleep hygiene behaviour, but there is no cut-off to label categories of sleep 15 hygiene engagement. This instrument demonstrates acceptable internal consistency (α = 16 [/fig] [fig] d = 0: 45) and moderate to large increases in sleep quality (d = 0.65). Pre-post correlations 19 were based on preliminary data taken from a trial targeting and measuring changes in 20 physical activity and sleep, 36 which showed correlations of r = 0.57 and r = 0.60 for physical 21 activity and sleep quality, respectively; therefore a pre-post correlation of 0.60 was assumed 22 in the current study. Assuming an alpha of 0.025 (due to measuring two primary outcomes; 23 MVPA and sleep quality), power of 0.80, a moderate effect size (d = 0.45 for physical 24 activity; d = 0.65 for sleep) and a pre-post correlation of 0.60, a total of 60 participants per 25 [/fig] [fig] 8 9: Study sponsorship, funding and organisation 10This study was supported in part by funds from a Future Leader Fellowship from the National 11Heart Foundation of Australia awarded to MJD (ID 100029) as well as a Vanguard Grant 12 from the National Heart Foundation of Australia awarded to MJD (ID 100629). [/fig] [fig] 10: will fulfil this role, in the case of BM's temporary illness or absence. BM, MJD, ATR and 11 RCP contributed to the development of study materials, MJD, CV and WJB have contributed 12 to the conceptual design of the trial and all authors (BM, RCP, ATR, CV, WJB and MJD) 13 contributed to the writing of the protocol and will be involved in the interpretation of results, 14 the evaluation of the trial and dissemination of study findings. 15 16 [/fig] [fig] Figure 1: Social cognitive factors related to physical activity and sleep hygiene Flow of participants in the Synergy Study 4 [/fig] [fig] Figure 2 5, Figure 3: Sleep hygiene log items Screenshots of app screens for self-monitoring and feedback relative to goals 6 D, Ricci C, Leitzmann MF. Associations of objectively assessed physical activity 9and sedentary time with all-cause mortality in US adults: the NHANES study. [/fig] [fig] Figure 3: Screenshots of app screens for self-monitoring and feedback relative to goals 209x148mm (300 x 300 DPI) [/fig] [fig] 14 15: Depression, anxiety, and stress 16The effect of changes in physical activity and sleep on participants' severity of depression, 17 anxiety and stress symptoms will be assessed using the Depression-Anxiety-Stress Scale 18 (DASS-21). The DASS-21 is reported to have satisfactory levels of internal consistency for 19 its total scale (r = 0.93) as well as for its individual scales for depression (r = 0.88), anxiety (r 20 = 0.82) and stress (r = 0.90). 60 In addition, DASS-21 scores will be examined as a review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open [/fig] [fig] 5: Severity Index (ISI) is a valid and reliable instrument for measuring insomnia severity.6 It can be used to classify individuals as having no insomnia (0-7), sub-threshold 6 insomnia (8-14), moderate clinical insomnia(1)(16)(17)(18)(19)(20)(21) or severe clinical insomnia (22-28). This 7 index will measure the proportion of the sample with potentially severe, yet undiagnosed 8 insomnia symptoms. Whilst assessing the severity of sleep problems and the level of 9 dissatisfaction with sleep a person can experience, the ISI also captures the extent to which 10 the consequences of sleep problems manifest themselves in everyday life, for example "To 11 what extent do you consider your sleep problem to interfere with your daily functioning (e.g. 12 daytime fatigue, mood, ability to function at work/daily chores, concentration, memory, 13 mood, etc.) currently?". Across a total of 7 items, responses are scored on a is a further indicator of poor sleep health. It will be measured using the 18 Epworth Sleepiness Scale (ESS), which assesses daytime sleepiness. This scale has 19 demonstrated high internal consistency (Crohnbach's alpha = 0.88) and good reliability (r = 20 0.82)66 and consists of 8 items that depicts various situations in which a person could 21 experience dozing off (e.g., while sitting and reading or watching TV). Items are summed to 22 calculate a total score from 0 to 24 with higher scores indicating higher levels of review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open [/fig] [fig] 18: social cognitive factors in the context of other health behaviours (i.e., physical activity, 19 diet). 74 76 Each scale will query each of the following nine sleep hygiene practices: (1) 20 avoiding caffeinated beverages (coffee, tea, energy drinks, etc.) in the late afternoon or right 21 before bedtime, (2) avoiding nicotine right before bedtime, (3) avoiding alcohol right before 22 bedtime, (4) exercising regularly, (5) reducing stress levels, (6) reducing the impact of noise 23 and nuisance in the bedroom, (7) keeping sleep and wake times consistent, (8) avoiding 24 daytime naps and (9) avoiding the use of technological devices (e.g., phone, TV, laptop, etc. [/fig] [fig] 20 21: Study sponsorship, funding and organisation 22This study was supported in part by funds from a Future Leader Fellowship from the National 23Heart Foundation of Australia awarded to MJD (ID 100029) as well as a Vanguard Grant 24 from the National Heart Foundation of Australia awarded to MJD (ID 100629). 25Page 41 of 63 For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open [/fig] [fig] 22: will fulfil this role, in the case of BM's temporary illness or absence. BM, MJD, ATR and 23 RCP contributed to the development of study materials, all authors (BM, RCP, ATR, CV, 24 WJB and MJD) have contributed to the conceptual design of the trial and the writing of the 25 Page 42 of 63 For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open [/fig] [fig] Figure 2: Sleep review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open [/fig] [table] Table 1: Overview and content of message-based support service [/table] [table] Table 3: Overview of outcome measures and assessment time points [/table] [table] 68: perceived behavioural capability, 69 10 outcome expectations and expectancies, 68 70 environment, 71 social support, 72 implementation 11 [/table] [table] Table 4: Social cognitive factors related to physical activity and sleep hygiene behaviours [/table] [table] 60: 2011;13(4):e126. doi: 10.2196/jmir.1923 [published Online First: 2012/01/03] 48 For peer review only -http://bmjopen.bmj.com/site/about/guidelines.xhtml BMJ Open [/table]

Association of parental characteristics and emotion regulation in children and adolescents with and without psychopathology: A case-control study Association of parental characteristics and emotion regulation in children and adolescentsSupplementAs gender and age of the children seem to influence ER and parenting behavior, we calculated the different analyzes with gender and age as supplemental variables in an explanatory fashion and reported this in S1-File. Regarding gender differences and age no specific hypotheses were made because research concerning age and gender is inconsistent. # Results ## Hypothesis 1 As matching led to a significantly smaller sample size, we performed the analyses with the original sample (n = 229) in an exploratory fashion to check for the influence of age and gender. As groups were no longer dependent but differed in age, we used a MANCOVA with gender as a supplemental factor and with age as a covariate. Using Pillai's trace there was a significant effect for group =10.39, p <.001.) but not for the covariate age (V = .01, F(2,223) = .74, p = .478). No effect for gender (V = .004, F(2,223) = .45) p = .639) and no interaction effect gender*group (V = .003, F(2,223) = .37 , p = .692) was found. Post hoc ANOVA revealed a significant effect for reappraisal (F(1,224))= 6.85, p = .009). There was also found a significant effect for suppression (F(1,224) = 11.33, p =.001). Including the additional factors didn't change the results. ## Hypothesis 2 As matching led to a significantly smaller sample size and to shed light on the influence of age and gender of the children on the parenting behavior, we performed the analyses with the original sample in an exploratory fashion again. As groups were no longer dependent but differed in age, we used a MANCOVA with age as covariate and gender as supplemental factor. The groups did not differ in their reaction also in the original sample (n = 229), introducing gender as a supplemental factor and controlling age as a covariate. Using Association of parental characteristics and emotion regulation in children and adolescents 1 Pillai's trace there was no significant effect neither for group (V = 0.006, F(2,223) = 0.63, p =.531) nor for the covariate age (V = 0.001, F(2,223) = 0.01, p = .910) nor for gender (V = .01, F(2,223) = 1.38, p = .254) nor for the interaction effect group*gender (V = .003, F(2,223) = .37, p =.629). ## Hypothesis 3 reappraisal To test the third hypothesis, we added to the original analysis the factors age and gender as two supplemental predictors. 139 cases could be included like in the original analysis, in two separate hierarchical multiple regressions (reappraisal and suppression). As there were significant deviations from normality in the variable mental health (zkurtosis =12,18; p <0.001; zskewness = 11,75, p <0.001), a bootstrapping procedure using 1,000 samples was used in the hierarchical multiple regressions. As shown in S1 Table for reappraisal, the first model step including factor age was not significant F(1,137) = .61, p = .437, adjusted R 2 = .003, ΔR² = .004. The second step, including gender was also not significant (F2,136) =1.33, p = .268) adjusted R 2 = .005, ΔR²=.02. The third step parents' reaction to their child's negative emotions was significant, F(4, 134) = 3.75, p = .006, adjusted R 2 = .074, ΔR².=.08 The fourth model step including parents' ER was also significant, F(6,132) = 3.38, p = .004 adjusted R 2 =.094, ΔR²=.03. The fifth step including of parents' mental health remained significant, F(7,131) = 22.88, p = .008, adjusted R 2 = .087, ΔR² =.001. However, no additional variance could be explained by mental health (see S1 . S1 Table shows the standardized and unstandardized regression coefficients with bootstrapped confidence intervals. Note. N = 139. CI = Confidence interval (based on 1,000 bootstrapped samples); LL = lower limit; UL = upper limit; z-age = mean standardized score on age; z-CCNES = mean zstandardized score on the Coping with Children's Negative Emotions Scale; z-ERQ = mean zstandardized score on the Emotion Regulation Questionnaire; z-GSI = mean z-standardized score on the Global Severity Index. ## S1 table. unstandardized (b) and standardized (β) regression coefficients for each ## Predictor in a hierarchical regression model predicting reappraisal in children ## Suppression A second hierarchical multiple regression was calculated to predict suppression based on age on step 1, gender on step 2, parents' reactions to negative emotions of their children at Step 3, emotion regulation in parents at Step 4, and mental health at step 5. Once again, a bootstrapping procedure based on 1,000 samples was applied. The first factor age was Note. N = 139. CI = Confidence interval (based on 1,000 bootstrapped samples); LL = lower limit; UL = upper limit; z-age = mean standardized score on ag; z-CCNES = mean zstandardized score on the Coping with Children's Negative Emotions Scale; z-ERQ = mean zstandardized score on the Emotion Regulation Questionnaire; z-GSI = mean z-standardized score on the Global Severity Index; [fig] significant F 1 ,: 137) = 23.61, p≤.001, adjusted R² = .14 , ∆R² = .19. Including the next four steps no additional variance could be explained (Step2 gender: (F(2,136) = 11.83, p ≤ .001, adjusted R² = .14, ∆R² =.001, step 3: F(4, 134) = 5.89, p ≤ .001, adjusted R² = .12, ∆R² = .001. Including emotion regulation strategies of parents did not significantly change the explained variance, F(6, 132) = 4.28, p = .001,. adjusted R² =-.12, ∆R² =.01. Finally, including mental health of parents did not increase the explained variance, F(7, 131) = 3.61, p = 001), adjusted R² =-.02, ∆R² =. 01. S2 [/fig]

Examining the Potential of Developing and Implementing Use of Adiponectin-Targeted Therapeutics for Metabolic and Cardiovascular Diseases Cardiometabolic diseases encompass those affecting the heart and vasculature as well as other metabolic problems, such as insulin resistance, diabetes, and non-alcoholic fatty liver disease. These diseases tend to have common risk factors, one of which is impaired adiponectin action. This may be due to reduced bioavailability of the hormone or resistance to its effects on target tissues. A strong negative correlation between adiponectin levels and cardiometabolic diseases has been well-documented and research shown that adiponectin has cardioprotective, insulin sensitizing and direct beneficial metabolic effects. Thus, therapeutic approaches to enhance adiponectin action are widely considered to be desirable. The complexity of adiponectin structure and function has so far made progress in this area less than ideal. In this article we will review the effects and mechanism of action of adiponectin on cardiometabolic tissues, identify scenarios where enhancing adiponectin action would be of clinical value and finally discuss approaches via which this can be achieved. ## Structural features of adiponectin Circulating adiponectin is primarily derived from adipose tissue, with lower-level expression detected in several other peripheral tissues (1-4). Adiponectin monomers are oligomerized within adipocytes prior to secretion and circulate as low molecular weight (LMW) medium (MMW) and high molecular weight (HMW) forms. Extensive post-translational modifications play a critical role in adiponectin forming these oligomeric complexes. In particular, the disulfide bond formed via Cys39 (human)/Cys36 (mouse) and the hydroxylation, glycosylation occurred on lysine residues (lys68, lys71, lys80, lys104) are essential for adiponectin to form the hexameric and oligomeric complexes, respectively (5, 6). The full-length adiponectin (fAd) oligomers can be cleaved by neutrophil elastase to liberate the carboxy-terminal globular domain (gAd) which itself possesses physiological activity (7, 8). ## Adiponectin receptors and signaling Adiponectin action is mediated primarily through adiponectin receptors 1 (AdipoR1) and 2 (AdipoR2), which are non-G-protein-coupled receptors containing seven transmembrane domains, but with an internal N-terminus and external Cterminus (9). Both adiponectin receptors are rather ubiquitously expressed although some variations do occur, such as more abundant AdipoR1 expression in skeletal muscle and higher AdipoR2 expression in liver (9, 10). In vitro experiments in C2C12 myocytes utilizing siRNA demonstrated that suppression of AdipoR1 reduced gAd binding while AdipoR2 suppression primarily reduced fAd binding, and their respective downstream signaling and functional effects (9). The functional roles of adiponectin receptors have been examined in transgenic or knockout mouse models of AdipoR overexpression created by different research groups. Yamauchi et al. reported undetectable levels of adiponectin specific binding and action in AdipoR 1 and 2 double-knockout mice leading to glucose intolerance and insulin resistance in these animals. Both AdipoR1-null and AdipoR2-null mice exhibited similar phenotypes with both strains showing increased adiposity and insulin resistance (11). A consistent phenotype of insulin resistance was observed in AdipoR1 deficient mice (12, 13), however studies in which AdipoR2 was deleted have reported opposing phenotypes in terms of glucose tolerance and susceptibility to diet-induced insulin resistance . Adiponectin binding to AdipoRs initiates a cascade of downstream signaling through the interaction of AdipoR to intracellular adaptor proteins (15) with APPL1 (adaptor protein containing pleckstrin homology domain, phosphotyrosine binding domain, and leucine zipper motif 1) acting as the primary adaptor protein mediating the metabolic effects of adiponectin (16). Adiponectin stimulation results in the binding of APPL1 to the cytoplasmic domain of AdipoR1 and AdipoR2 via the phosphotyrosine binding (PTB) and coiled coil (CC) domain of APPL1 (17). Subsequent translocation of LKB1 to the cytosol as well as calcium release from the endoplasmic reticulum through phospholipase C activates calcium/calmodulin-dependent protein kinase . AMPK activation is the central mechanism whereby adiponectin stimulates metabolic effects (6, 7, 10, 13, 17, 18, 20-26), induces NO-dependent vasodilation, inhibits the production of reactive oxygen species (ROS), and modulates mTOR signaling. In addition to AMPK activation, several AMPK-independent pathways exists whereby adiponectin is able to regulate insulin sensitivity, inflammation, glucose uptake, and ceramidase activity (27). ## Physiological effects of adiponectin and implications in cardiometabolic disease The diverse physiological functions of adiponectin in metabolic and cardiovascular tissues has significant implications in health and disease states. Multiple studies have established primarily beneficial effects of adiponectin on the regulation of metabolism, immunity, inflammation, cardiac remodeling, vasculature control and cancer (16, 28-30). The anti-diabetic actions of adiponectin include insulin sensitizing and insulin mimetic actions in liver and skeletal muscle, as well as protection against beta cell destruction in the pancreas (31). In addition to this, increased glucose transport and GLUT4 translocation by adiponectin in skeletal muscle is regulated by AMPK or p38 MAPK activation (17). Adiponectin increases fatty acid oxidation through PPARα enhanced expression of target genes in the liver (20, 22, 23) or through increased mitochondria biogenesis in skeletal muscle (13). The cardioprotective effects of adiponectin can be attributed in part to effects on cardiac metabolism, apoptosis, autophagy and hypertrophy (32). Additional cardioprotection is mediated by the anti-inflammatory, antioxidant, and vasorelexant properties of adiponectin as well as its ability to inhibit atherogenesis (31). Initial in vivo studies examining the effect of adiponectin on atherosclerosis demonstrated that adenovirusmediated overexpression of adiponectin (33) and gAd treatment (23) in apolipoprotein (apo) E-deficient mice resulted in reduced atherosclerosis. Systematic review and meta-analysis of human clinical trials suggests an important role of adiponectin in the development of atherosclerosis, as hypoadiponectinemia was associated with early carotid artery atherosclerosis lesions in healthy and metabolic disease populations (34). It should be noted that this association was weak (34) and not consistent across all studies (35) but in vitro experiments as well as animals studies have reported data supporting the anti-atherogenic properties of adiponectin. Adiponectin inhibits multiple steps involved in the development of atherosclerotic lesions including the reduction of macrophage lipid accumulation, inhibition of macrophage to foam cell formation, suppression of proinflammatory cytokines release and lymphocyte migration, inhibition of leukocyte and endothelial cell interaction, and suppression of vascular smooth muscle proliferation through the inhibition of atherogenic growth factors (31, 36). In the early development of atherosclerosis, adiponectin has been demonstrated to inhibit monocyte-macrophage migration, thus reducing the attachment of monocytes to injured endothelial cells and the formation of macrophage foam cells (37, 38). In addition to this, adiponectin can downregulation scavenger receptor A (SR-A) and acyl-coenzyme A: cholesterol-acyltransferase 1 (ACAT1) expression, both of which are important regulators of macrophage lipid accumulation and foam cell formationAdiponectin can also inhibit foam cell formation by modulating lipid metabolism and cholesterol efflux in macrophagesand altering the activity of enzymes (i.e., heptaic lipase, lipoprotein lipase) involved in the catabolism of lipoproteins. The effects of adiponectin on foam cell formation is mediated through an adiponectin-AdipoR1/2-APPL1 axis in macrophages. Thus, adiponectin is a critical factor regulating the development of atherosclerosis which accounts for its beneficial effects on cardiometabolic diseases. Dysregulation of cardiac energy metabolism is an important feature seen in early stages of heart failure. Cardiomyocyte contractile energy in the healthy heart under aerobic conditions is derived from fatty acids (∼70%) and glucose (∼30%) and this balance is perturbed under conditions of cardiac stress, such as under ischemic conditions. Our groupand othershave shown that adiponectin can regulate cardiac energy metabolism. Both globular and fulllength adiponectin stimulation led to increased glucose uptake and metabolismvia the activation of AMPK, IRS1, and Akt1 in primary neonatal rat cardiomyocytes. The effect of adiponectin on cardiomyocyte glucose uptake is dependent on Rho/ROCK regulated actin cytoskeleton remodeling which colocalized APPL1 to actin filaments. Similar results were observed in primary adult rat cardiomyocytes with adiponectin significantly enhancing insulin stimulated Akt phosphorylation and glucose uptake. The effect of lipotoxicity on cardiac insulin signaling was examined in adiponectin knockout mice and a significant reduction in insulin stimulated Akt signaling was observed in high fat fed mice compared to chow fed group and adiponectin replenishment was able to reverse the defect in insulin signaling. Adiponectin treatment increased the expression of fatty acid transporter protein-1 and induced the translocation of CD36 to the outer cell membrane in primary neonatal and adult rat cardiomyocytes, respectively, resulting in increased fatty acid uptake. Adiponectin, via AdipoR1-APPL1 signaling, regulates fatty acid β-oxidation in primary rat cardiomyocytes and isolated working heart through the activation of AMPK leading to acetyl coenzyme A carboxylase (ACC) phosphorylation and enhanced fatty acid oxidation. These results suggest that adiponectin isoforms regulate cardiac metabolism and function leading to more efficient utilization of glucose and fatty acids. Adiponectin attenuates cardiomyocyte apoptosis seen in heart failure through several different signaling pathways. Adiponectin treatment was protective against stress-induced apoptosis through Akt-dependent signalingin HL-1 cardiomyocytes. In cultured H9c2 cells and adiponectin-null mice, globular adiponectin attenuated hypoxia/reoxygenationinduced apoptosis through the reduction of reactive oxygen species. Adiponectin stimulation of ceramidase activity plays a vital role in cell survival through ceramide degradation and production of sphingosine 1-phosphate (S1P) both of which protected primary mice neonatal ventricular cardiomyocytes from palmate-induced apoptosis. Our group has recently demonstrated the importance of ceramidase activity in the cardioprotective effects against reactive oxygen species (ROS) and apoptosis. High fat diet (HFD) in adiponectin knockout mice increased myocardial total triglyceride, ceramides, and sphingosine-1-phosphate (S1P) compared to chow-fed animals with adiponectin replenishment resulting in significant reduction in S1P intracellular content. When these conditions were simulated in an in vitro model of lipotoxicity, palmitate treatment significantly increased S1P intracellular concentrations in H9c2 cells. Addition of a synthetic adiponectin receptor agonist, AdipoRon, to palmitate-treated H9c2 cells reduced intracellular concentrations while simultaneously increasing secretion of S1P which was consistent with attenuation of palmitate-induced ROS production and cell death. Thus, in vivo and in vitro results suggests that through the activation of cardiac ceramidase activity, adiponectin increases the conversion of ceramide to S1P which is secreted to exert autocrine/paracrine cardioprotective effects. Regulation of cardiomyocyte autophagy is now recognized as an important mechanistic component of the cardioprotective effects of adiponectin. Examination of myocardial autophagy in an in vivo animal model of oxidative stress induced by chronic angiotensin II (Ang-II) treatment showed increased LC3II protein expression in the left ventricle which was significantly higher in adiponectin knockout vs. wild-type mice, this most likely resulting from a block of autophagic flux. H 2 O 2 induced autophagic cell death in isolated adult rat ventricular myocytes through AMPK/ERK activation, mTOR inhibition, and increased expression of authophagic protein. This led to autophagasome accumulation and adiponectin pre-treatment ameliorated the effect of oxidative stress on autophagy. Cardiac autophagy is a dynamic process which is activated in response to stress but can become inhibited in chronic pathological conditions. In studies examining the effect of HFD on autophagy in wild-type and adiponectin knockout mice, a compensatory elevation in autophagy in response to HFD was lacking in Ad-KO mice. Aortic banding to induce pressure overload cardiac dysfunction resulted in activation of cardiac autophagy in wild-type mice but this change was deficient in adiponectin-null mice. Inhibited autophagy was also observed in adiponectin knockout mice following lipopolysaccharide challenge compared to wild-type mice which had upregulated LPS-induced autophagy through AMPK-mTOR-ULK1 dependent signaling. In vitro studies with H9c2 cells, revealed an important role of adiponectin in the stimulation of autophagy flux in cardiomyocytes at multiple steps within the autophagy pathway. In adiponectin deficient mice, the normal autophagic response of cardiomyocytes to stressors is inhibited and in vitro data suggests that adiponectin replenishment can correct this abnormal response. Collectively, in vitro and in vivo animal studies provide convincing evidence of multiple mechanisms in which adiponectin can enhance cell survival and metabolism through the regulation of autophagy. Cardiac hypertrophy can be a normal physiological response which occurs secondary to pressure overload, however under specific circumstances hypertrophy can become pathological and lead to cardiac dysfunction (32). Adiponectin has been demonstrated to protect against pathological cardiac hypertrophy in both in vitro and in vivo experiments. Multiple studies have reported exaggerated pathological cardiac hypertrophy in adiponectin knockout mice under experiment models of pressure overload (61-63) with adiponectin replenishment resulting in reduced pathologic hypertrophy. Experiments in isolated neonatal rat ventricular myocytes treated with angiotensin II to induce hypertrophy illustrated that the primary anti-hypertrophic mechanism of adiponectin occurred through AdipoR1-APPL1-AMPK activation resulting in suppression of nuclear factor kappa-B-induced hypertrophic growth signaling. These studies suggest that adiponectin can protect against cardiac hypertrophy and lack of adiponectin can lead to development of more severe pathological hypertrophy. In summary, evidence to date illustrates that the cardioprotective effects of adiponectin are mediated through effects on multiple cell types (e.g., endothelial cells, vascular smooth muscle cells, cardiomyocytes, fibroblasts, macrophages) involved in cardiac metabolism, survival and remodeling. ## The role of adiponectin as a biomarker Adiponectin circulates at high concentrations in humans and rodentsand hypoadiponectinemia is associated with metabolic syndrome across different ethnic groups (34,. Adiponectin levels in humans are inversely related to BMI and fat mass with reduced adiponectin mRNA expression, HMW adiponectin secretion and total serum levels observed in obesity, insulin resistance, T2DM, CVD, and metabolic syndrome (34,. Human studies have reported an inverse correlation between adiponectin levels and the risk of cardiovascular morbidity and mortality. Hypoadiponectinemia has been associated with early carotid artery atherosclerosis lesions in humans (34, 74), while higher serum adiponectin levels are correlated with favorable cardiovascular risk profiles in both males and females. Higher adiponectin levels were also associated with a reduced risk of developing type 2 diabetes and the subsequent risk of cardiovascular events in a large population of healthy patients. Consistent with these cardioprotective effects are studies demonstrating an association between persistent hypoadiponectinemia following acute myocardial infarct with increased risk of future major adverse cardiovascular events. In contrast, multiple studies have reported increased adiponectin levels in patients with advanced cardiovascular disease. In a prospective study of men and women without initial diagnosis ischemic heart disease or HF, higher adiponectin levels was directly correlated with increased risk of heart failure. The EXAMINE trial found a direct relationship between adverse cardiovascular outcomes and higher adiponectin levels in type 2 diabetic patients with recent acute coronary syndrome. In patients with chronic heart failure, high adiponectin levels were associated with an increased risk of mortality. Clinical studies in older populations demonstrated a positive association between adiponectin levels and mortality in patients with heart failure. The paradoxical association of adiponectin levels in relation to cardiovascular disease was further illustrated by observations from the Copenhagen City Heart Study which reported a positive relationship between high adiponectin and decreased risk factors for CVD in patients without initial CVD disease. This same study also found a positive direct correlation between adiponectin levels and all-cause mortality and major adverse cardiovascular events. This adiponectin paradox has been proposed to occur secondary to adiponectin resistance, a hypercatabolic state or in response to elevated natriuretic peptides which are found in advanced CVD. In particular, both human and animal studies have suggested that adiponectin resistance as a consequence of AdipoR1 downregulation may be present in severe CVD and thus hyperadiponectinemia is a compensatory mechanism. The wasting theory proposes that the hypercatabolic state in severe heart failure leads to increased adiponectin which is consistent with the inverse relationship between adiponectin and fat mass. Supporting this theory are observations showing hyperadiponectinemia only in the presence of cachexia in heart failure patients. Cardiac natriuretic peptides, atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP), are important mediators in the crosstalk between heart and adipose tissue. This was demonstrated in transgenic mice models where whole body as well as adipose tissue specific deletion of the NP clearance receptors, resulting in enhanced ANP action, protected mice from diet induced obesity and insulin resistance. In human cultured adipocytes, ANP stimulated lipolysis (90) and promoted the "browning" of white adipose tissue through enhanced energy expenditure via upregulation of mitochondrial biogenesis, respiration and UCP1 expression. Unpublished data from our group suggest that defective cardiac autophagy leads to reduced ANP-mediated inter-organ crosstalk leading to impaired glycemic control. Increased natriuretic peptides release is an indicator of cardiac stress and is used as a clinical biomarker of heart failure. Both ANP and BNP were found to enhance the expression of adiponectin mRNA and secretion from primary human adipocytes. This observation was reproducible in human studies with infusion of ANP resulting in increased total and HMW adiponectin concentrations in healthy menand patients with chronic heart failure. Clinical studies have also provided evidence for a positive, independent association between adiponectin levels and BNP in healthy subjectsand in men with ischemic heart disease. Concomitantly elevated proBNP was associated with elevated adiponectin levels observed in patients who developed heart failure in an 8.5 years follow-up prospective study of subjects without initial diagnosis of heart disease. Thus, both ex vivo and in vivo analyses have provided evidence supporting the idea that hyperadiponectinemia in advanced CVD can be driven by elevated natriuretic peptides. Overall, the clinical value of adiponectin as a biomarker remains highly promising, especially for metabolic and cardiovascular diseases. However, published findings suggest that clinical interpretation of circulating adiponectin levels must be done in the context of factors, such as age, gender, and severity or stage of CVD. ## Adiponectin as a biotarget Research studies to date have proven the significant potential of adiponectin as a biotarget for the modulation of metabolic and cardiovascular disease. However, the exogenous administration of recombinant adiponectin has proven to be difficult due to the challenges associated with producing stable multimeric recombinant adiponectin isoforms. Further complicating the process of establishing a therapeutic dose is the high endogenous concentration and the relative short in vivo half-life of adiponectin. The therapeutic success of adiponectin thus lies in approaches aimed at enhancing endogenous levels of expression as well as strategies to target adiponectin signaling and its downstream effector pathways. ## Current knowledge and future approaches for how adiponectin can be targeted therapeutically ## Increase circulating or local levels As summarized in, overcoming the reduction in circulating adiponectin levels observed in disease states, such as obesity and diabetes has potential to be beneficial. Lifestyle interventions are an effective strategy to increase circulating adiponectin levels. Interventions, such as exercise, caloric restriction, pharmacological drugs, or gastric bypass leading to weight loss have consistently shown a positive effect on adiponectin levels. Weight loss induced by a combination of lifestyle and pharmacological (phentermine/topiramate) interventions resulted in an increase in adiponectin levels in patients with metabolic syndrome. Circulating adiponectin levels significantly increased following gastric bypass and sleeve gastrectomy in obese women and this was negatively correlated with body weight and waist circumference. Both gastric bypass and very low calorie diets led to improved adiponectin levels in obese type 2 diabetic subjects. Exercise training leading to weight loss in overweight, obese and diabetic subjects was associated with increased adiponectin levels. These findings support the idea that sustained weight reductions through lifestyle modifications can enhance adiponectin levels. Thiazolidinediones (TZD), prescribed for the treatment of diabetes, are perhaps the most extensively characterized regulator of adiponectin expression. TZDs, such as pioglitazone and rosiglitazone increase adiponectin expression through the activation of proliferator-activated receptor gamma (PPARγ). Pioglitazone treatment for 16-weeks increased adiponectin levels in an obese population of Chinese subjects with diabetes and this correlated with improved insulin secretion and insulin sensitivity. Clinical studies in subjects across different ethnic, age and metabolic disease groups have consistently observed increased adiponectin levels with pioglitazoneor rosiglitazone (116) treatment. Importantly, TZDs up-regulate both total expression and HMW oligomers content in both in vitro as well as in vivo animal and human studies (3,. However, the adverse side effects which have unfortunately blighted use of TZDs (119, 120) limit their utility as a go-to agent to increase adiponectin levels. Specific dietary supplements have been recognized as regulators of adiponectin expression. Evidence from in vitro and in vivo studies suggests that vitamin E can upregulate adiponectin expression, also through the activation of PPARγ. Treatment with γ-and α-tocopherol, vitamers of vitamin E, enhanced adiponectin expression in 3T3-L1 adipocytes and miceand was able to ameliorate the suppressive effects of TNF-α on adiponectin expression in 3T3-L1 adipocytes. Both fish oil (123) and omega-3 (124, 125) supplementation increased adiponectin levels in human studies. St. John's Wort (Hypericum perforatum)and Groundsel Bush (Baccharis halimifolia) extract (127) induce 3T3-L1 adipocyte differentiation leading to enhanced adiponectin expression. Additional supplements that have been shown to increase circulating adiponectin in humans and animals include grape-seed extractgreen tea extract, walnuts, α-linolenic acid (135), resveratrol, and Radix Astragali isolated compounds (astragaloside II and isoastragaloside I). Targeting the renin-angiotensin aldosterone system through angiotensin-converting enzyme inhibitor (ACEi) and angiotensin receptor blockers (ARBs) have consistently increased adiponectin levels in humans through PPARγ activated adiponectin gene transcription and enhanced adipogenesis. The ACEi inhibitor temocapril also significantly increased adiponectin expression in patients with essential hypertension. Ex vivo experiments with human omental (OM) preadipocytes from healthy women showed significantly higher adiponectin mRNA expression in adipocytes differentiated in the presence of ARBs vs. TZDs. Finally, it was suggested that enhanced adiponectin expression was specific to PPARγ ligand ARBs (e.g., irbesartan, telmisartan) via studies in 3T3-L1 adipocytes and ex vivo epididymal fat from Zucker fa/fa rats (142). Incretins [e.g., glucagon-like peptide 1 (GLP-1), GLP-1 analogs (e.g. liraglutide)] and pharmacological agents used to increase incretin bioavailability [e.g., dipeptidyldipeptidase 4 (DPP4) inhibitors] upregulate adiponectin expression in 3T3-L1 adipocytes (143) as well as in clinical trials. Evidence from systematic review and meta-analysis of randomized controlled trials suggests that statins can increase adiponectin concentrations despite its negative effect on insulin sensitivityand risk for developing diabetes mellitus. Empagliflozin, a diabetes pharmacological agent that works through the inhibition of sodium-glucose cotransporter-2, was been reported to increase adiponectin levels in mice through an unknown mechanism. Together, these studies suggest that multiple pathways can regulate circulating adiponectin expression either through direct stimulation of gene expression or through mechanisms that enhance adipogenesis and insulin sensitivity. ## Alter level (or localization) of adiponectin receptors or signaling pathway proteins Regulation of AdipoR expression and downstream signaling effector proteins are clearly prime candidates which have been targeted to enhance adiponectin action and several compounds have been identified as possible regulators of adiponectin receptor expression. PPAR agonists have been reported to enhance AdipoR expression in various cell types. In epididymal white adipose tissue (WAT) of male KKAy mice, AdipoR1 and AdipoR2 expression was upregulated following treatment with the PPARα agonist Wy-14,643 (149). AdipoR2 but not AdipoR1 mRNA expression was increased by PPARα and PPARγ agonist in primary human and THP-1 macrophages. Rosiglitazone increased both AdipoR1 and AdipoR2 mRNA in isolated adult rat ventricular cardiomyocytes. In humans, pioglitazone treatment increased both AdipoR1 and AdipoR2 mRNA in muscle biopsies from type 2 diabetic subjects. Telmisartan, an ARB with selective PPARγ activity, was observed to correct the reduced ventricular cardiomyocytes AdipoR2 and aortic AdipoR1 in diabetic rat to comparable levels as control animals. Metformin, a first-line pharmacotherapy for treatment of diabetes, is a potent activator of AMPK with insulin sensitizing effects. Studies in ZDF rats demonstrated that metformin can upregulate AdipoR1 and AdipoR2 receptor expression in muscle and AdipoR1 in WAT. In addition to effects on adiponectin levels, exercise training in animals and humans is associated with enhanced AdipoR expression. Studies in obese and diabetic animal models consistently report upregulation of skeletal muscle AdipoR1 expression in response to different exercise programs. In humans, AdipoR1 and AdipoR2 expression in skeletal muscle increased in response to endurance exercise programs. Collectively, these findings further support the benefit of lifestyle interventions in enhancing adiponectin action. ## Receptor agonists-what's available and future developments AdipoR agonistshave been an intense focus of pharmaceutical drug development programs in metabolic and cardiovascular diseases (20, 158). Ligand binding receptors are of course recognized as the logical targets for activating/inhibiting that signaling pathway. Given the difficulties in producing biologically active adiponectin and optimizing the exact dosage and route of administration for this recombinant protein, designing agonists to activate adiponectin receptor-mediated downstream signaling is a highly desirable strategy to maximize adiponectin's beneficial effects. Several small molecules or short peptides have been discovered for this purpose since Kadowaki's group identified AdipoR1 and AdipoR2 (9). ## Adp355/adp399 The C-terminal of adiponectin (residues 105-254) that forms gAd is well-established to induce potent biological effects in various studies (21, 159). By screening 66 small peptides overlapping each other by 10 amino acids and covering the entire sequence of the globular domain of adiponectin, Otvos et al. discovered that the peptide sequence between residues 149-166 retained biological activity similar to gAd (160). A patent was filed (US9,073,965) indicating the potential of developing this sequence into a receptor agonist. After additional pharmacological modifications and structure-function assays, a short peptide named ADP355 (H-DAsn-Ile-Pro-Nva-Leu-Tyr-DSer-Phe-Ala-DSer-NH2), was formed acting as an active adiponectin receptor agonist. Follow up in-vitro activity assays including cell proliferation assay and western blot to monitor the activation of key signaling molecules (AMPK, AKT, STAT3, ERK1/2 etc.) in various cancer cell lines, revealed effects of ADP355 in the concentration range 100 nM−10 uM comparable to or exceeding those of gAd. A bolus injection of 5-50 mg/kg ADP355 displayed excellent stability and minimum toxicity in-vivo. Furthermore, a 1 mg/kg/day ADP355 treatment via intraperitoneal (i.p.) injection for 28 days significantly inhibited the growth of human breast cancer xenograft in mouse (161). With the initial success, Otvos et al. continued to explore ways to pharmacologically enhance the agonist activity of ADP355. In considering adiponectin normally circulates in its multimeric forms, the second generation of the peptide, a linear branched dimer (ADP399) and an octapeptide (ADP400), was formed. The dimeric peptide ADP399 exhibits almost 20-fold increased cellular activity in comparison to the monomeric peptide ADP355. However, surprisingly, at the similar concentration, ADP400 acted as an antagonist rather than an agonist to AdipoRs. ## Adiporon AdipoRON, a synthetic small molecule that can be administered orally, is the most well-studied AdipoR agonist currently available. In 2013, via screening chemical library compounds provided by the Open Innovation Center for Drug Discovery in University of Tokyo, Kadowaki's group successfully identified AdipoRON as the most potent agonist for AdipoR's. During the screening assay, the phosphorylation of AMPK, the key targeted signaling molecule for adiponectin, was used as the readout for evaluating the activity of all chemical compounds in the screen. In C2C12 myotubes, comparing to the treatment of native adiponectin protein, AdipoRON induced a comparable and dose-dependent phosphorylation of AMPK within the concentration of 5-50 uM. AdipoRON was identified as agonist for both AdipoR1 and AdipoR2. An intravenous injection of AdipoRON, in the dose of 50 mg/kg, induced the phosphorylation of AMPK in both skeletal muscle and liver of wt mice, however, this phosphorylation was abolished in AdipoR1 and AdipoR2 double knockout mice. One key advantage of a small molecule receptor agonist in the pharma industry is oral administration. To test the therapeutic potential of AdipoRON in alleviating metabolic disorders, 50 mg/bodyweight of AdipoRON was given to diet induced obese mice via oral administration for 8 days. AdipoRON effectively improved insulin sensitivity and restored glucose homeostasis via the activation of AdipoR1-AMPK-PGC1α and AdipoR2-PPARα signaling pathways. AdipoRON treatment also mimicked adiponectin's established anti-diabetic effectsand ability to enhance cellular capacity for mitigating oxidative-stress, enhancing lipid/glucose oxidation in mitochondria, anti-inflammatory responses, lifeprolonging effect, anti-cancer effects, procell survival and anti-apoptotic effect, neuronal-, reno-, and cardio-/vascular-protective effects. However, exciting AdipoRON research in animal models has not been translated to establishment of a drug for human use and the search continues for additional small molecule AdipoR agonists which have little or no toxicity. ## Osmotin Osmotin, a plant protein, that was found to be structurally similar to adiponectin, can also induce the phosphorylation of AMPK in C2C12 myotubes. Interestingly, the adiponectin receptor, AdipoR1, is the mammalian homolog of the osmotin receptor, PHO36. Based on these observations, it was proposed that osmotin could function as an agonist for AdipoR1. Indeed, an osmotin peptide with nine residues (CTQGPCGPT) was synthesized and its molecular interaction with AdipoR1 was modeled in silico. Functional analyses revealed similar biological effects, evaluated as the induction of IL-6 production in synovial fibroblasts, of fAd (5 ug/ml) and osmotin peptide (5 ug/ml). Studies with in-vivo, in-vitro treatment of osmotin also revealed its adiponectin's memetic effect toward obesity, diabetes and related fatty liver, cardiovascular diseases. The activation of AdipoRs mediated downstream signaling pathways including AdipoRs/PPARαand AdipoR1/PI3K/AKT (183) upon osmotin treatment, further confirmed the agonist activity of osmotin peptide to AdipoRs, in particularly to AdipoR1. Interestingly, a group of Korean scientists led by Dr. Kim MO have conducted series of studies and revealed the neuroprotective effect of osmotin, in particular to Alzheimer disease. Firstly, they shown the preventive effect of osmotin on amyloid beta-induced synaptic deficits, Aβ accumulation, β-secretase expression and tau phosphorylation via reduced phosphorylation of PI3K, Akt, and GSK in mice. Secondly, they identified AdipoR1/TLR4/NFκB, AdipoR1/AMPK/SIRT1/SREBP2 signaling, and AdipoR1 interfered Nogo-receptor 1 (Ng1) signaling are three key pathways that responsible for osmotin diminished neuroinflammation and Aβ accumulation while improved neurodegenerative disease related, synaptic deficits, cognitive impairment, memory loss and long-term potentiation. Lastly, an osmotin preloaded magnetic nanoparticles demonstrated a novel drug delivery approach for the treatment of Alzeimer. ## Others Additional AdipoR activating small molecules have also been studied as potential therapeutic agents. Via an in silico approach, peptide Pep70 was identified as a potential AdipoR1 agonist in protecting against fibrosis. PEGylated BHD1028 was discovered and formulated for the treatment of diabetes. Others identified that a short region of adiponectin protein Nterminus (6, 15-35) can act as agonist to AdipoRs and activate the downstream signaling pathways to promote cell viability and proliferation, thus maintaining pancreatic beta cell mass and preventing the development of diabetes. Another orally active AdipoR agonist named6-C-b-D-glucopyranosyl-(2S,3S)-(+)-5,7,39,49-tetrahydroxydihydroflavonol (GTDF) was identified and its biological effects characterized, followed by identification of a 13-amino acid residue segment located in the collagen domain of adiponectin (ADP-1) as another potent AdipoR1 agonist. The corresponding functional studies have revealed that GTDF can bind to both AdipoRs, with a preference for AdipoR1, and activate associated signaling pathways to improve metabolic health, including glucose uptake, lipid profile, beta cell survival, reduced steatohepatitis and the browning of adipose tissue. Many efforts have been invested in identifying and optimizing AdipoR agonists as a class of therapeutic drugs, however, none of them has reached the stage of being adopted in clinical practice yet. This may due to the interpersonal variants in endogenous adiponectin and adiponectin receptor expression levels and the complicated cross-reactive molecular networks formed around its signaling pathway. Besides, it is also important to realize that the development of adiponectin resistance in certain disease states, possibly due to the reduced expression or altered post-translational modification of adiponectin receptors or adaptor proteins, such as APPL1, could also significantly affect the effectiveness of AdipoR agonists (10,. However, with recent advances in high throughput and high content drug discovery technologiesand the establishment of AdipoR crystal structure to facilitate rational drug design, it is possible to expect further refinement of small molecule AdipoR agonists for the treatment of metabolic and cardiovascular diseases. ## Future considerations a personalized approach to maximizing effectiveness of adiponectin therapeutics There clearly is huge potential for implementing use of adiponectin-based therapeutics, and when the time comes we must also make progress in stratifying ways to better deploy their use. Here, we list several possible directions that could be follow to maximize health impact of future advances in adiponectinrelated drug discovery. ## Crosstalk with the microbiome As our understanding of the bio-effects of adiponectin has developed during the last decade, so too has knowledge of the important role that gut microbiota plays in metabolic health. Like adiponectin, targeting microbiota has also been suggested to have therapeutic value in treating metabolic syndrome. A large number of studies in rodents and humans have now validated the concept that insulin resistance condition could be improved upon receiving a more healthy donor's intestinal microbiota via a procedure called fecal microbiota transplantation (FMT). Interestingly, it was found that expression of adiponectin can also be modulated by specific changes in gut microbiota composition. FMT, especially of those that are enriched in Lactobacillus NK6 colony (very similar to Lactobacillus taiwanensis strain BCRC 17755) can induce adiponectin expression from gut epithelial cells. Indeed, it has been proposed that activation of adiponectin signaling can also mediate the beneficial physiological effects of Lactobacillus. The important role of gut microbiota in mediating drug absorption and metabolism has now been recognized. Specifically, one study identified more than five phase-I metabolites and many possible glucuronic acid conjugated phase-II metabolites generated upon AdipoR agonist AdipoRON treatment. However, whether these metabolites are generated from microbe or host mediated drug metabolism, and whether there is any functional significance related to those metabolites are still under investigation. Thus, future studies focused on developing adiponectin-based therapies must include considering the dynamic interaction among gut microbiota, host metabolism and adiponectin based drug metabolism. ## Artificial intelligence (ai) assisted drug development and use The analysis of pharmacokinetics and pharmacodynamics on adiponectin based drug metabolism is often long and costly, yet artificial intelligence using machine learning algorithms are now able to assist this process. An example for AI assisted drug development is analysis of the hepatic toxicity effect of terbinafine, an oral antifungal agent. Nearly 20 years after the initial safety watch regarding its hepatic toxicity effect, a machine learning algorithm trained with large dataset of known metabolic pathways identified what no human could previously: the detailed 2-step metabolic processes that led to the breakdown of terbinafine into its toxic metabolite, TBF-A. Using an AI assisted computational approach could help in forming a comprehensive understanding of metabolism for new adiponectin-mimetic small molecules and thus assist in making logical predictions of potential pharmacological considerations. FIGURE 3 | Enhanced adiponectin therapeutics via precision medicine upon personalized response prediction. Accumulation of big data will allow algorithm-based prediction of those individuals most likely to benefit from therapeutic agents to either increase adiponectin levels or directly mimic adiponectin action, thus resulting in more efficient and improved health care outcomes. ## Precision medicine to maximize impact of adiponectin-based therapeutics Interpersonal variants in factors including the expression level of adiponectin and adiponectin receptors, the largely diverse life style (such as food composition, exercise frequency, sleep quality), the stage at which individuals are located along the course of disease progression, ultimately determine the individualized response to potential therapeutic interventions. The reciprocal interplay between microbiota and adiponectin action has also created personal variables in drug efficiency. All the above listed factors played important role on the yet not so successful translational trials on adiponectin based drug development. In order to design the most effective treatment plan according to all such variants, the concept of precision medicine is important. Several pioneering studies have illustrated the power of personalized profiling in understanding and capturing individualized characterization under the same clinical phenotype. For example, unique groups of small molecules were identified as being characteristic for each individual and that they differed from the group mean. These individualized datasets can serve as early predictive signatures for disease warning and allow specific preventive treatments to be planned accordingly in order to delay the occurrence of diseases. These pioneering studies provide an excellent guide in establishing a concept that will be applicable for future studies focused on adiponectin as a personalized therapeutic target. In conclusion, merging what we know about adiponectin in disease pathophysiology, and mechanisms of action with new understandings of how utilizing AI can personalize medicine and improve outcomes, it is hoped that adiponectin-based therapeutics can be tailored as the best-in-class approach for managing metabolic health in the future . # Author contributions All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. # Acknowledgments

Clinical outcomes of helical tomotherapy for super-elderly patients with localized and locally advanced prostate cancer: comparison with patients under 80 years of age We investigated the clinical outcomes of helical tomotherapy in 23 patients aged ≥80 years with localized and locally advanced prostate cancer and compared the results with data from 171 patients under 80 years. All patients received helical tomotherapy in our hospital between September 2009 and October 2012. The median follow-up periods were 35 months in the aged group and 34 months in the younger group. The median prescribed dose in helical tomotherapy was 78 Gy in 39 fractions (range, 72-78 Gy). The 3-year overall survival and biochemical relapse-free rates were 92% and 96% in the aged group and 99.4% and 97.3% in the younger group, respectively. There was no significant difference between the two groups in the biochemical relapse-free rates. The 3-year cumulative incidences of late Grade 2 or higher rectal toxicity and urinary toxicity were 13% and 4.8% in the aged group and 7.0% and 1.2% in the younger group, respectively. There was no significant difference between the aged group and the younger group in the cumulative incidence rates of rectal toxicity or urinary toxicity. No patients exhibited Grade 4 or higher toxicity, and all patients improved with conservative therapy. Helical tomotherapy in superelderly patients with localized and locally advanced prostate cancer had good biochemical control rates without severe late toxicity. Definitive helical tomotherapy may be the treatment of choice for patients with localized and locally advanced prostate cancer, even in those older than 80 years of age. # Introduction Prostate cancer is one of the most common malignancies in the USA [bib_ref] Cancer statistics, Siegel [/bib_ref] , Europe [bib_ref] Cancer incidence and mortality patterns in Europe: estimates for 40 countries in..., Ferlay [/bib_ref] and Japan [bib_ref] Cancer incidence and incidence rates in Japan in 2007: a study of..., Matsuda [/bib_ref]. The incidence of prostate cancer is increasing among the aging population in Japan [bib_ref] Cancer incidence and incidence rates in Japan in 2007: a study of..., Matsuda [/bib_ref]. The US National Cancer Institute database lists a median age at diagnosis of 68 years, and 71.2% of deaths due to prostate cancer occur in men aged over 75 years. With the dramatic aging of populations and increasing life expectancy (LE), especially in developed countries [bib_ref] Rapid increase in Japanese life expectancy after World War II, Sugiura [/bib_ref] [bib_ref] Trends in European life expectancy: a salutary view, Leon [/bib_ref] , further increases in the number of patients with prostate cancer are expected. Guidelines for the management of elderly patients (usually defined as aged 65 or older) with prostate cancer have been established based on accumulating clinical data [bib_ref] Do older men benefit from curative therapy of localized prostate cancer?, Alibhai [/bib_ref] [bib_ref] Radical prostatectomy versus watchful waiting in early prostate cancer, Bill-Axelson [/bib_ref] [bib_ref] Overtreatment of men with low-risk prostate cancer and significant comorbidity, Daskivich [/bib_ref]. The National Comprehensive Cancer Network (NCCN) guidelines emphasize that estimating LE is important when choosing the therapeutic strategy for localized and locally advanced prostate cancer. Definitive radiation therapy is considered for intermediate-and high-risk groups of prostate cancer patients with LE of <10 years. Alibhai et al. [bib_ref] Do older men benefit from curative therapy of localized prostate cancer?, Alibhai [/bib_ref] developed a Markov state transition model to evaluate LE and quality-adjusted LE (QALE) in men older than 65 years with localized prostate cancer. They concluded that radical prostatectomy and definitive radiation therapy improved the LE and QALE up to age 75 years for moderately differentiated tumors (Gleason 5-7), and curative therapy improved the LE and QALE up to age 80 years for poorly differentiated tumors ( . Recent studies show that elderly men are more likely to be diagnosed with higher-risk prostate cancer than younger men [bib_ref] Contemporary risk profile of prostate cancer in the United States, Shao [/bib_ref]. As the elderly population grows, the number of patients with prostate cancer who require definitive radiation therapy is expected to increase accordingly. However, the Cancer of the Prostate Strategic Urologic Research Endeavor database indicates that men aged over 75 years were more likely to be treated conservatively and to die from prostate cancer [bib_ref] Contemporary risk profile of prostate cancer in the United States, Shao [/bib_ref] [bib_ref] Impact of age at diagnosis on prostate cancer treatment and survival, Bechis [/bib_ref]. One reason that the appropriate treatment is not selected for elderly prostate cancer patients may be the lack of data on the clinical outcomes of definitive therapy for elderly patients with prostate cancer. Hence, clinical outcomes in super-elderly patients (80 years and older) with localized and locally advanced prostate cancer treated with intensity-modulated radiation therapy (IMRT) in our hospital were investigated. The data were compared with those from prostate cancer patients under the age of 80 years. In addition, the correlation between the dosimetric parameters and late toxicity was analyzed. # Materials and methods ## Patient characteristics A total of 23 patients aged 80 years or older with T1c-T4 non-metastatic prostate cancer (according to the International Union Against Cancer TNM Classification of Malignant Tumors, 7th edition) were treated with IMRT in our hospital between September 2009 and October 2012. All patients were included in the present study. To evaluate the clinical outcomes in super-elderly patients who were 80 years old or above, data were compared with those from 171 patients under the age of 80 years with intermediate-risk, high-risk and castration-resistant prostate cancer (CRPC) who were treated around the same time in our hospital. The institutional review board of our hospital approved this study. [fig_ref] Table 1: Patient characteristics [/fig_ref] shows the patient characteristics in the present study. The median ages were 81 years old (range, 80-87) in patients aged 80 years and above (the aged group) and 71 years old (range, 46-79 years) in patients under the age of 80 years (the younger group). All patients were classified using the D'Amico risk group classification [bib_ref] Biochemical outcome after radical prostatectomy, external beam radiation therapy, or interstitial radiation..., D&apos;amico [/bib_ref]. Eight patients were classified as intermediate-risk and 15 as high-risk in the aged group; 59 patients were classified as intermediate-risk and 112 patients were classified as high-risk in the younger group at the time of diagnosis. Eight patients in the aged group and five patients in the younger group were diagnosed with CRPC before IMRT initiation because of a continuous increase in the serum levels of prostate-specific antigen (PSA), despite the castrated levels of serum testosterone (<50 ng/dl). Therefore, five patients were intermediate-risk, 10 were high-risk, and eight had CRPC in the aged group, and 58 patients were intermediate-risk, 108 patients were high-risk, and five patients had CRPC in the younger group at the start of IMRT. The median periods from the start of androgen deprivation therapy (ADT) to diagnosis with CRPC were 73 months (range, 14-138 months) in the aged group and 37 months (range, 22-61 months) in the younger group. The median follow-up periods after IMRT were 35 months (range, 19-53 months) in the aged group and 34 months (range, 11-61 months) in the younger group. Written informed consent was obtained from all patients prior to treatment. Thoraco-abdominal computed tomography (CT, Aquilion LB, Toshiba Medical, Otahara, Japan) and pelvic magnetic resonance images (MRIs, Vantage Titan and Pianissimo, Toshiba Medical, Otahara, Japan) were performed for staging before the start of IMRT in all patients. Whole-body bone scintigraphy was also performed before IMRT initiation for patients with high-risk prostate cancer and CRPC. ## Imrt using helical tomotherapy IMRT is a powerful tool for improving the quality of the delivered dose distribution in external beam radiation therapy, and this therapy reduces late rectal toxicity in high-dose external beam radiation therapy for prostate cancer [bib_ref] Localized volume effects for late rectal and anal toxicity after radiotherapy for..., Peeters [/bib_ref]. The TomoTherapy Hi-Art system (Accuray Inc., Sunnyvale, CA, USA) is a radiation delivery system that combines dynamic IMRT and an image-guided radiation therapy system [bib_ref] History of tomotherapy, Mackie [/bib_ref] [bib_ref] Tomotherapy: a new concept for the delivery of dynamic conformal radiotherapy, Mackie [/bib_ref]. All patients with prostate cancer were treated with IMRT using helical tomotherapy (TOMO) in the present study. ## Ct simulation and target delineation Patients were placed in the supine position with a universal fixation device (ESFORM, Engineering System Co., Nagasaki, Japan) to immobilize the lower legs and reduce set-up error. Axial images (3-mm slices) were acquired using a 16-row multi-detector CT (Aquilion LB, Toshiba Medical, Otahara, Japan) for planning CT. Pelvic MRI at 3-mm thickness was performed, and T2-weighted images were fused to the planning CT images to delineate target volumes. Contouring of target volumes and normal structures was performed on the Focal treatment planning system, version 4.3.1 (Focal Eindhoven, Netherlands). The clinical target volume (CTV) was defined as the entire prostate and proximal seminal vesicles. The CTV was defined as the entire prostate and all of the seminal vesicles in patients with T3b disease. The planning target volume (PTV) encompassed the CTV with a 5-mm margin in the bilateral, craniocaudal and anterior directions, and there was a 3-mm margin in the posterior direction. The rectum was contoured as a solid structure defined by the outer wall from the anal canal to the rectosigmoid junction. The bladder was also contoured as a solid structure defined by the outer wall. Two or more radiation oncologists examined all contoured structures to provide consistency in defining the target volumes. The contours created in the treatment-planning system were transferred to the TomoTherapy Hi-Art treatment-planning system, v4.0, and the TOMO plans were generated. ## Planning of tomo The PTVs primarily received 78 Gy in 39 fractions in the present study. Three cases in which the intestines were close to the PTV received 72 Gy in 36 fractions to reduce toxicity. The prescription dose was defined as the minimum dose delivered to 95% of the PTV (D95%). The maximum tolerated dose in the PTV was limited to <105% of the prescription dose. Rectal dose-volume constraints limited V65 Gy ( percentage of the rectum volume receiving at least 65 Gy) to ≤17%, V40 Gy to ≤35% and V22 Gy to ≤60%. The bladder dose-volume constraints limited V65 Gy to ≤25% and V40 Gy to ≤50%. The appropriate dose constraints were implemented for inverse planning procedures. The following treatment parameters were used to generate the TOMO plans: field width of 2.48 cm, modulation factor of 2.0, and pitch of 0.287. To reduce the dose to critical organs, protective measures such as rectal blocking structures were appropriately added in the TOMO planning. All optimization procedures were performed until the both the PTV and other organs' dosevolume constraints were achieved. The entire optimization was restarted when constraints were violated. A fine grid (2.7 mm × 2.7 mm) was used for the final calculation process after all constraints were satisfied. ## Daily treatments of tomo Image-guided radiation therapy was performed daily in all patients. Acquired CT images using mega voltage CT (MVCT) were superimposed onto the treatment plans. The patient's position was adjusted according to prostate matching before each treatment. Patients had a tube inserted or were encouraged to defecate when their rectums were dilated for daily MVCT and were re-examined on MVCT. Androgen deprivation therapy ## Follow-up after tomo and data collection A urologist and a radiation oncologist conducted patient follow-ups at 3-month intervals for the first 3 years after TOMO and at intervals of 3-6 months thereafter. PSA values were evaluated in each follow-up examination. Biochemical relapse was defined as the nadir PSA level plus 2 ng/ml. CT images were collected once per year after TOMO to assess any metastatic progression. Late toxicity was evaluated according to the Common Terminology Criteria for Adverse Events, version 3.0. Patients with suspected rectal bleeding underwent endoscopic examinations, and late rectal toxicity was confirmed. For patients aged 80 years and above, dose-volume histogram (DVH) parameters were collected from each treatment plan on the TomoTherapy Hi-Art treatment-planning system to clarify the effect of late toxicity. Patients were divided into two groups: those with any late toxicity and patients without late toxicity. We compared the DVH parameters and complications, such as diabetes mellitus and conditions requiring the use of anti-coagulation therapy, in these two groups. The present study analyzed the PTV volume, rectal volume, maximum rectal dose, and minimal radiation doses for the most irradiated rectal volumes of 2 cc (Rectum D2 cc), V70 Gy, V60 Gy, V50 Gy, V40 Gy and V30 Gy to assess effects on late rectal toxicity. Similarly, the PTV volume, bladder volume, maximum bladder dose, Bladder D2 cc, V70 Gy, V60 Gy, V50 Gy, V40 Gy and V30 Gy were analyzed to assess effects on late urinary toxicity. ## Statistical analyses The overall survival time, biochemical relapse-free time, and cumulative occurrence rates of late toxicity were estimated using the Kaplan-Meier method. Comparisons of the overall survival time, biochemical relapse-free time, and cumulative occurrence rates of late toxicity were assessed by Log-rank analysis. Comparisons of the clinical and dosimetric factors between the two groups based on toxicity were assessed using Fisher's exact test, the Wilcoxon signed-rank test, and Welch's unpaired t-test. Multivariable logistic regression analysis was performed for factors that previously appeared to be associated with the risk of late rectal toxicity using the t-test (P < 0.05). Significant differences were assessed using two-sided tests with P < 0.05. All statistical analyses were performed using Statistical Package for the Social Sciences (SPSS) software, version 16.0 (SPSS, Chicago, IL, USA). # Results Overall survival and biochemical relapse-free rates shows the overall survival rates and biochemical relapse-free rates. In the cohort of patients aged 80 years and older, the 3-year overall survival and biochemical relapse-free rates were 92% and 96%, respectively. Two patients had died by the final follow-up. One patient had exhibited biochemical relapse at 6 months and had died of metastatic prostate cancer at 34 months after TOMO initiation, and the other patient died of gastric cancer at 42 months after the initiation of TOMO, with no evidence of prostate cancer recurrence. The patient who died of metastatic prostate cancer after TOMO was in the high-risk group. All eight CRPC patients had no evidence of . Overall survival rates and biochemical relapse-free rates after TOMO. Solid lines show the overall survival rates, and dashed lines indicate the biochemical relapse-free rates after TOMO. Blue lines depict the rates for patients 80 years and older, and green lines illustrate rates for patients younger than 80 years old. There was a significant difference in the overall survival rates. disease at the final follow-up. In the cohort of patients younger than 80 years of age, the 3-year overall survival and biochemical relapsefree rates were 99.4% and 97.3%, respectively. There was a significant difference between the aged group and the younger group in the overall survival rates (P < 0.014). There was no significant difference between the two groups in the biochemical relapse-free rates. [fig_ref] Table 2: Maximal late toxicities in all cases [/fig_ref] shows the maximal late toxicities in the present study. No Grade 4 or 5 late toxicity was observed in any of the patients. Of the patients aged 80 years and older, Grade 3 late toxicity was observed in one patient, who developed massive rectal bleeding and required a blood transfusion 22 months after the initiation of TOMO. Grade 2 late toxicity was observed in three patients: two patients with rectal bleeding and one patient with urinary retention. None of the patients with Grade 2 or 3 toxicity required surgical intervention (e.g. laser coagulation), and these patients improved with conservative therapy (e.g. oral drug administration). Grade 1 late toxicity was observed in five patients; four patients had slight rectal bleeding and four had urinary frequency. Some patients exhibited both rectal and urinary events. No late toxicity was observed in the remaining 14 patients. Of the patients younger than 80 years, Grade 3 late toxicity was observed in three patients, and Grade 2 late toxicity was observed in 10 patients. None of these patients required surgical intervention. shows the cumulative occurrence rates of developing late Grade 2 or higher rectal or urinary toxicity after TOMO. In the aged group, the 3-year cumulative incidences of rectal and urinary toxicity were 13% and 4.8%, respectively. In the younger group, the 3-year cumulative incidence rates of rectal and urinary toxicity were 7.0% and 1.2%, respectively. There was no significant difference between the aged group and the younger group in either cumulative incidence rates of rectal toxicity or urinary toxicity. ## Late toxicity The effects of patient characteristics and DVH parameters on late rectal toxicity [fig_ref] Table 3: Patient characteristics and mean values of the rectal dosimetric parameters in patients... [/fig_ref] shows the effects of patient characteristics and DVH parameters on late rectal toxicity in patients aged 80 years and older. Patients with late rectal toxicity had significantly higher mean rectal values of V40 Gy, V50 Gy, V60 Gy and V70 Gy. The most significant difference was observed for V70 Gy (P < 0.005). Multivariable logistic regression analysis for the factors that were associated with the risk of late rectal toxicity using the t-test indicated that none of the factors was significantly correlated in the analysis (data not shown). The effects of patient characteristics and DVH parameters on late urinary toxicity [fig_ref] Table 4: Patient characteristics and mean values of the urinary dosimetric parameters in patients... [/fig_ref] shows the effects of patient characteristics and DVH parameters on late urinary toxicity in the cohort of patients aged 80 years and older. There were no significant differences between the patients with and without late toxicity in any patient characteristics or DVH parameters. # Discussion In the patients aged 80 years and older, only one patient died of prostate cancer metastases 34 months after TOMO, and all other patients were alive without recurrence after TOMO at the final follow-up (median follow-up time: 35 months). There was a significant difference in the overall survival rates between the cohort of patients younger than 80 years old and those who were 80 years and older. However, this difference was due to the death of two patients in the aged group, one of whom died from gastric cancer. It is unlikely that the difference in ages resulted in a difference in the therapeutic effect of TOMO. Additionally, the 3-year cumulative incidences of late Grade 2 or higher rectal toxicity and urinary toxicity in the present study were 13% and 4.8%, and no patients with Grade 4 or higher toxicity were observed in the present study. The incidences of toxicity were not significantly different in the older group from those of patients younger than 80 years old. Additionally, these incidences of toxicity were similar to those reported in previous studies of high-dose IMRT for prostate cancer, most of . Cumulative occurrence rates of late Grade 2 or higher rectal and urinary toxicities after TOMO. The solid lines show the cumulative occurrence rates of rectal toxicity, and dashed lines indicate the cumulative occurrence rates of urinary toxicity. Blue lines illustrate rates for patients 80 years and older, and green lines show the rates for patients younger than 80 years old. There was no significant difference in the cumulative incidence rate or rectal or urinary toxicity. which were undertaken in patients aged 80 years or younger [bib_ref] Outcome and toxicity for patients treated with intensity modulated radiation therapy for..., Vora [/bib_ref] [bib_ref] Intensitymodulated radiation therapy for prostate cancer: late morbidity and results on biochemical..., De Meerleer [/bib_ref] [bib_ref] Long-term outcome of high dose intensity modulated radiation therapy for patients with..., Zelefsky [/bib_ref] [bib_ref] Results and DVH analysis of late rectal bleeding in patients treated with..., Someya [/bib_ref] [bib_ref] Toxicity and efficacy of three dose-fractionation regimens of intensity-modulated radiation therapy for..., Manabe [/bib_ref] [bib_ref] Transitioning from conventional radiotherapy to intensity-modulated radiotherapy for localized prostate cancer: changing..., Yamazaki [/bib_ref]. These findings indicate that TOMO for patients who are 80 years and older with localized and locally advanced prostate cancer may be acceptable and beneficial. The burden of prostate cancer is expected to increase dramatically with the exponential aging of populations and increasing LE, especially in developed countries [bib_ref] Rapid increase in Japanese life expectancy after World War II, Sugiura [/bib_ref] [bib_ref] Trends in European life expectancy: a salutary view, Leon [/bib_ref]. Definitive radiotherapy based on the careful consideration of an individual's prostate cancer risk profile should be considered, even for patients over 80 years of age. Recently, Tomita et al. [bib_ref] Preliminary analysis of risk factors for late rectal toxicity after helical tomotherapy..., Tomita [/bib_ref] reported a relationship between the dosimetric parameters and late rectal toxicity for localized prostate cancer patients treated with TOMO. They demonstrated that rectal V60 Gy and V70 Gy, and the maximum dose to the rectum, were significantly higher in the ≥ Grade 2 toxicity group than the ≤ Grade 1 toxicity group. There was a similar trend in that patients with late rectal toxicity had significantly higher mean values of rectal V40 Gy, V50 Gy, V60 Gy and V70 Gy. Hence, we recommend the use of stringent dose-volume constraints in middle to high doses to the rectum in TOMO for prostate cancer to reduce rectal toxicity. No significant correlation was observed between the dosimetric parameters of the bladder and late urinary toxicity in the present study. More than one-third (8 of 23) of the patients aged 80 years or older in the present study had CRPC, probably because most superelderly patients were administered ADT without local treatment as the primary treatment in practice [bib_ref] Antiandrogen monotherapy: a new form of treatment for patients with prostate cancer, Kolvenbag [/bib_ref] [bib_ref] Bicalutamide 150 mg: a review of its use in the treatment of..., Wellington [/bib_ref]. After the disease developed into CRPC, such a patient would be recommended to receive radiotherapy, such as TOMO. However, several recent reports have demonstrated inadequate efficacy of radiotherapy for CRPC, and the biochemical relapse-free survival rates in these reports were not satisfactory [bib_ref] External beam radiotherapy for clinically localized hormone-refractory prostate cancer: clinical significance of..., Ogawa [/bib_ref] [bib_ref] External beam radiotherapy for clinically node-negative, localized hormone-refractory prostate cancer: impact of..., Akimoto [/bib_ref]. The use of this conventional radiotherapy technique, which used lower prescribed doses than the present study, might explain these unsatisfactory outcomes. In fact, the use of high-dose radiotherapy with TOMO in all eight patients with CRPC in the present study eliminated biochemical recurrence until the final follow-up. These findings indicate that optimized high-dose radiotherapy may achieve better outcomes, even for CRPC. The most recently released NCCN guidelines do not define established treatment strategies for CRPC patients without signs of lymph node or distant metastases. Androgen receptor status remains positive in most patients who develop CRPC, and secondary hormone therapy such as anti-androgen, ketoconazole, steroids, diethylstilbestrol, or other estrogens, are other options for CRPC treatment [bib_ref] Antiandrogen withdrawal alone or in combination with ketoconazole in androgen-independent prostate cancer..., Small [/bib_ref] [bib_ref] Prospective, multicenter, randomized phase II trial of the herbal supplement, PC-SPES, and..., Oh [/bib_ref]. However, no randomized clinical trial has demonstrated benefits in terms of survival rates. Moreover, secondary hormone therapy is often not used for super-elderly patients in clinical practice due to an increased risk of adverse effects, such as diabetes, cardiovascular complications and osteoporosis. The present study indicated that TOMO achieved advantageous outcomes with low toxicities in super-elderly patients with CRPC. Therefore, high-dose IMRT using TOMO is one option for the salvage treatment of super-elderly patients with node-negative, localized CRPC. The present study has several limitations, including the retrospective nature of the study, the small number of patients analyzed, and the short follow-up period. In addition, all patients who received IMRT with TOMO exhibited good performance status without severe comorbidities. Rockwood et al. [bib_ref] A brief clinical instrument to classify frailty in elderly people, Rockwood [/bib_ref] reported that senior adult patients who were dependent in terms of daily living activities had a shorter survival. Furthermore, Guzzo et al. [bib_ref] Prediction of mortality after radical prostatectomy by Charlson comorbidity index, Guzzo [/bib_ref] reported that the non-prostate cancer-specific mortality rate of patients with localized prostate cancer treated with radical prostatectomy was higher than the prostate cancer-specific mortality rate, and the Charlson index [bib_ref] A new method of classifying prognostic comorbidity in longitudinal studies: development and..., Charlson [/bib_ref] , which evaluates comorbidities, was the strongest predictor of death from causes other than prostate cancer. Taken together, when we treat super-elderly patients with prostate cancer using any treatment, including TOMO, it is necessary to thoroughly examine the suitability of the treatment through assessing the performance status, comorbidities and risk of prostate cancer. Active surveillance is considered the best option for patients with low-risk prostate cancer. In fact, patients with low-risk prostate cancer were not included in this study. The NCCN guidelines recommend starting treatment in most patients who have a Gleason grade of 4 or 5 on repeat biopsy, cancer in a larger number or greater extent of prostate biopsies, or a PSA doubling time of less than 3 years. ADT might be commonly used as a treatment option for patients with prostate cancer that has progressed after active surveillance in the clinical setting, especially in the elderly. However, Lu-Yao et al. [bib_ref] Survival following primary androgen deprivation therapy among men with localized prostate cancer, Lu-Yao [/bib_ref] reported that there was no survival benefit in patients receiving ADT compared with observation alone in an analysis of 19 271 elderly patients who did not receive definitive local therapy for clinical stage T1-T2 prostate cancer. ADT should not be used in routine practice for elderly patients with low-risk prostate cancer. Additionally, some studies have reported an association between ADT and an increased risk of diabetes [bib_ref] Diabetes and cardiovascular disease during androgen deprivation therapy for prostate cancer, Keating [/bib_ref] , cardiovascular morbidity [bib_ref] Influence of androgen suppression therapy for prostate cancer on the frequency and..., D&apos;amico [/bib_ref] , and bone fractures [bib_ref] Risk of fracture after androgen deprivation for prostate cancer, Shahinian [/bib_ref]. These ADT-related adverse effects could be fatal in super-elderly patients. Although ADT is generally considered to be effective in combination with radiation therapy for patients with intermediate-and high-risk prostate cancer, further research is required to identify the suitable period of ADT for super-elderly patients with prostate cancer. Our findings show that TOMO may be beneficial in patients who are 80 years and older with localized and locally advanced prostate cancer. Further follow-up is required to determine long-term outcomes. [table] Table 1: Patient characteristics [/table] [table] Table 2: Maximal late toxicities in all cases [/table] [table] Table 4: Patient characteristics and mean values of the urinary dosimetric parameters in patients aged 80 years and older Age, bladder volume, PTV and dose-volume histogram parameters are represented as the means ± standard deviation. CTCAE v3.0 = The Common Terminology Criteria for Adverse Events version 3.0, PTV = planning target volume, no. = number, V dose = the percentage of the rectum at least covered by each dose, n.s. = not significant. [/table] [table] Table 3: Patient characteristics and mean values of the rectal dosimetric parameters in patients aged 80 years and older Age, rectal volume, PTV, and dose-volume histogram parameters are represented as the means ± standard deviation. CTCAE v3.0 = The Common Terminology Criteria for Adverse Events version 3.0, PTV = planning target volume, no. = number, V dose = the percentage of the rectum at least covered by each dose, n.s. = not significant. [/table]

Novel, disposable, self-inserted, vaginal device for the non-surgical management of pelvic organ prolapse: efficacy, safety, and quality of life Background: We evaluated a novel disposable, collapsible, ring-shaped vaginal device that is self-inserted within an applicator and removed with a string. The device was developed to overcome the drawbacks of existing ring pessaries for non-surgical pelvic organ prolapse management (POP).Methods:The primary objective efficacy endpoint of this prospective, interventional, multicenter, self-controlled, and home-use study was the proportion of subjects with improved staging on the Pelvic Organ Prolapse Quantification (POP-Q) scale. Subjective efficacy was assessed using the POP symptoms alleviation score. Safety was evaluated by recording the rate and incidence of adverse events (AEs) in a daily diary, and quality of life (QoL) was evaluated using the modified Pelvic Floor Impact (PFIQ-7) and Pelvic Floor Disability Index (PFDI-20) questionnaires.Results:A total of 94 usage cycles were observed in a group of 52 participants (mean age 60.2 ± 10.5 years, 81.1% postmenopausal) who used the device for 3558 days. Of these, 24 participants completed one usage cycle, 14 completed two usage cycles, and 14 completed three usage cycles with 28-45 days of ProVate use in each usage cycle. All patients experienced greater than two POP-Q stage reductions. The descent was completely reduced to POP-Q stage 0 in 97.8% of participants. The POP symptom alleviation questionnaire showed significant subjective efficacy (P < 0.0001). The modified PFDI-20 and PFIQ-7 scores also improved substantially (P < 0.0001 for both). There were 91 nonserious device-related AEs: 98.9% were mild and 87.9% anticipated, with no vaginal infection, and one case of urinary tract infection.Conclusion:The novel device substantially reduces prolapse and provides significant subjective POP symptom relief and QoL improvement, with minimal AEs. The device may enable women to self-manage their prolapse with a small, disposable device that minimizes self-touching and frequent dependency on the clinic.Trial registration: Clini cal. Trials. gov, NCT02 239133, posted September 12, 2014 (retrospectively registered). POP [bib_ref] Forecasting the prevalence of pelvic floor disorders, Wu [/bib_ref] , with 210,000-300,000 undergoing surgical treatments per year and the rest being managed with vaginal pessaries, mainly ring-shaped, or remaining untreated. Although data on the proportion of pessary users is scarce, a 1:1 ratio of pessary users to women with untreated POP can be assumed [bib_ref] Outcomes of observation as therapy for pelvic organ prolapse: A study in..., Gilchrist [/bib_ref]. Pessaries have been used in the non-surgical management of POP for decades; they are considered effective and safe. However, existing pessaries have substantial drawbacks, which limit their widespread use [bib_ref] Pessary use and management for pelvic organ prolapse, Atnip [/bib_ref]. Moreover, they are associated with a high rate of discontinuation, which exceeds 50% within 12 months [bib_ref] Predictors of successful ring pessary use in women with pelvic organ prolapse, Fatakia [/bib_ref] , with the major reasons being failure to retain the pessary and inability to insert and remove the device by the user, desire for another treatment modality (e.g., surgery), adverse events (AEs), and sexual disturbances. Existing ring pessaries are reusable only [bib_ref] Pelvic Organ Support Study (POSST): the distribution, clinical definition, and epidemiologic condition..., Swift [/bib_ref] and are large and intrusive, with diameters ranging from 54 to 110 mm. They are partially squeezed (reduced dimensions) during insertion but fully opened during removal, resulting in the most common AEdiscomfort and pain when pressing and widening the introitus. We assumed that more women than previously believed require or want non-surgical POP management, and that vaginal pessaries may be a viable option for them. However, they are hesitant to use them due to multiple difficulties, just as many healthcare providers (HCPs) are hesitant to recommend a treatment with so many complications [bib_ref] Pessary or surgery for a symptomatic pelvic organ prolapse: the PEOPLE study,..., Van Der Vaart [/bib_ref]. The huge gap between existing cumbersome pessary management and women's desire for a more pleasant, self-manageable, and comfortable POP control, including unhindered intercourse, necessitated the development of a new device that combined the benefits of a ring pessary with substantial reductions in, or elimination of, its major drawbacks. To develop a new treatment option, we conducted a literature search and HCPs surveys for understanding the most bothersome complaints related to existing ring pessaries and designed mitigation options to overcome each complaint [fig_ref] Table 1: Identified downsides of existing ring pessaries and possible mitigationsIdentified problems with currently... [/fig_ref]. Following this, we developed the ProVate device, which has a slender (28 mm) body, that transforms into a ring pessary with six sizes (61-91 mm) and is intuitively selfinserted/removed, similar to how a menstrual tampon is inserted and removed. ProVate is a small, disposable, flexible, and selfexpanding vaginal ring pessary that comes ready for use within an applicator and with easy removal after a collapse by pulling a string. Like existing rigid ring pessaries, ProVate was designed to function as a scaffold that lifts the prolapsed vaginal walls once in place. Expectedly, when pessaries of equal size are used, their intravaginal mechanical function should be equivalent. Furthermore, their objective and subjective efficacies and impact on the quality of life (QoL) are expected to be similar. However, with ProVate, fewer AEs as well as enhanced user experience and satisfaction are expected, allowing women to self-manage POP and their intimate behavior [bib_ref] Technical update on pessary use, Robert [/bib_ref]. The design of a prospective longitudinal home trial to assess objective and subjective efficacies, safety, QoL, and users' satisfaction was driven by the comparable functionality of ProVate and a ring pessary. The following were the trial's specific characteristics: - A single-arm trial in which each user served as her own control (demonstrating quantifiable POP comparisons before and during using the device through well-established performance indicators, such as the Pelvic Organ Prolapse (POP-Q) quantification scale [bib_ref] The standardization of terminology of female pelvic organ prolapse and pelvic floor..., Bump [/bib_ref] and validated QoL questionnaires [bib_ref] Responsiveness of the Pelvic Floor Distress Inventory (PFDI) and Pelvic Floor Impact..., Barber [/bib_ref]. - A longitudinal, hypothesis-driven, and statistically powered study where some participants used the device for more than one ~45-day usage cycle (e.g. 2-3 usage cycles). This allowed data collection from 94 usage cycles of ~45 days each and from users who repeatedly used the device during 2 & 3 consecutive usage periods for over 2 years. This design facilitated follow-up of users during longer periods, over long time span, with a larger number of evaluable participants (94 usage cycles in 52 partcipants). The study assumed that although use was moved into the homes and hands of laymen, the device's efficacy (prolapse stage and related symptoms) in reducing POP would remain high. It was also anticipated that AEs would remain low after insertion and removal in small dimensions of an easy-to-use disposable device for a short period of time. Women can replace the device as frequently as they wish, with limitations of up to 7 days per device, to ensure the low rate of AEs, as this complies with the guidelines of the Society of Obstetricians and Gynecologists of Canada for women who can comply with pessary self-care [bib_ref] Guideline No. 411: Vaginal Pessary Use, Harvey [/bib_ref]. # Methods This research was a prospective, interventional, multicenter, one-arm, open-label, self-controlled, home-use study. The study's objective was to confirm the efficacy and safety of the ProVate device for regular use. The study was performed following the ethical standards in the Declaration of Helsinki. Furthermore, the institution's ethics committee approved the research (Assuta-Maccabi Helsinki committee, app #2014038), after which each participant provided written informed consent. The study was conducted in three outpatient gynecology/urogynecology clinics in Israel between August 2014 Insertion Manual insertion only: either by a healthcare provider or by the user in the small amount of women who can self-care. Insertion by the user herself, within a disposable applicator, at her will, regardless of place and time. ## Removal Manual removal only in the large dimensions: either by a healthcare provider, or by the user in the small amount of women who can self-care Removal by the user herself, by pulling a string at her will, regardless of place and time. The device collapses to small dimensions for comfortable removal. Length of use Either every 3+ month by a healthcare provider, or every 1-7 days in the small amount of cases where the user is able to self-care. Each device should be used for a limited length of time, preferably up to a week. Adverse events Vaginal discharge, irritation, infection and foul smell are well described known adverse events. A disposable only device, with short usage duration, is expected to substantially reduce or eliminate adverse events. Dependency on the clinic A reusable device which is re-used for years, requires, in most cases, dependency upon clinic visits. No dependency upon clinic visits. Device is self-inserted, Irrespective of time and place. Other points Need to self-touch when able to self-care. Removal before intercourse is available only in women who can self-care. No need of self-touch. Device comes ready for use within a disposable applicator. Easy removal before intercourse. and June 2016. Symptomatic participants were recruited from the clinics' database or following advertisements. presents the study flow and device usage. After screening (visit 1), eligible participants were fitted with the correct size (visit 2) and underwent training on device use. Size confirmation was conducted at the study clinic following 2-3 days of home use (visit 3). Then, the subject was refitted if the size was either too small (causing expulsion) or too large (discomforting). During the usage period, participants were instructed to use as many devices as they wished for 1 to 7 days each, and to fill in a daily diary, documenting each device's length of use, functionality, and AEs. The home-use portion of the study (termed the usage period or usage cycle) began after visit 3 and lasted up to 45 days. Participants were instructed to use ProVate for at least 28 days within the 45 days. An ultrasound scan was conducted to estimate the post-void residual (PVR) urine before and while using ProVate. Participants were examined vaginally during each clinic visit by the same gynecologist/urogynecologist to assess the stage of prolapse (POP-Q scale) with or without the device and look for signs of infection, bleeding, and vaginal wall trauma. Similar ProVate models were tested in an iterative, consecutive fashion, including the final marketed version. Changes between device models were limited to the applicator, whereas the actual ProVate ring, which affects efficacy and safety, remained the same. Inclusion criteria included women aged 21-80 years with symptomatic sensation of vaginal prolapse, diagnosed with POP-Q stage 2-4 prolapse in one or more sites along the vaginal walls, ability to use both hands The ProVate Device with its various shapes during insertion and removal. The ProVate device is provided clean and individually wrapped. It is available for immediate vaginal insertion using a disposable applicator (a). During vaginal insertion, which is similar to inserting a menstrual tampon, the plunger is pushed, and the slender compacted device within the applicator gradually enlarges to become a ring (b). After fully pushing the plunger, the ring becomes fully deployed (c). The applicator then separates from the ring and is removed from the vagina for disposal, leaving the string available for later removal (d). The deployed ring may remain in the vagina for up to 7 days (e). A pull on the string collapses the ring into is its slender pre-insertion size for comfortable removal and disposal (f-h). (Source: ConTIPI Medical Ltd., with permission) Flow diagram of methodology comparing POP-Q results from the end of visit 5 with those of baseline and insert a device into the vagina, and the ability to retain a 61-to 91-mm pessary. Exclusion criteria included previous inability to accommodate tampons or vaginal pessaries; current participation in another clinical study; comorbid condition(s) or severe systemic diseases that could limit the subject's ability to participate in the study; pregnancy, suspected pregnancy, or intention to become pregnant during the study; abnormal vaginal bleeding in the previous 6 months; previous vaginal surgery during the preceding 3 months; severely atrophic vagina; existing vaginal or vulvar laceration; symptomatic vaginal or urinary tract infection, as determined by physical examination and lab results; recurrent urinary tract infections; and abnormal cervical cytology. The primary endpoint of this study was the proportion of participants who showed an improvement of at least 1 stage from baseline on the POP-Q scale on the fifth visit while using ProVate, as evaluated by per-protocol (PP) analysis. The analysis tested the null hypothesis that the proportion of participants with an improvement from baseline was <70%. The alternative hypothesis was that the proportion of participants with an improvement was ≥70%. The null hypothesis was tested using the exact binomial test. The secondary objective efficacy endpoint was the proportion of participants who were eventually evaluated as POP-Q stage 0 or 1 prolapse on the final visit. The secondary subjective endpoints included the improvement of POP symptoms (assessed by an authorcompiled POP symptoms alleviation questionnaire), improvement in QoL (assessed using the modified PFDI-20 and modified PFIQ-7 questionnaires), and participant satisfaction with the device (evaluated by an author-compiled questionnaire). Objective efficacy, or improvement in the prolapsed stage, was evaluated at all study visits using the POP-Q scale. Additionally, subjective efficacy was assessed using the POP symptoms alleviation score, which was developed and compiled by the authors to assess the change in POP-related complaints before and during treatment. Ten specific POP-related complaints were graded on a scale ranging from 0 to 4 (0 = no complaint at all to 4 = significant complaint), and scores from visit 1 (before using the ProVate) and visit 5 (while using the ProVate) were normalized to the 0-100 scale, analyzed, and compared. Changes in QoL were assessed using the applicable parts of the validated PFDI-20 and PFIQ-7 QoL questionnaires (i.e., only those questions that are pertinent to POP). In the modified PFIQ-20 score, 10 of the 20 questions in the original questionnaire were used to assess the burden of pelvic floor disorders due to specific inabilities. Possible scores range from 0 to 4, where 0 = not at all and 4 = very much. In the modified PFIQ-7 questionnaire, which quantifies the burden of various pelvic floor disorders on the ability to perform certain daily activities, only the seven questions related to the vagina or pelvis were incorporated, with possible scores ranging from 0 to 3, where 0 = not at all and 3 = very much. Results were normalized to a scale of 0-100. Participants were questioned regarding their satisfaction while using the device during the three specific usage steps: insertion, usage, and removal. At the end of the study (visit 5), complaints particular to the use of the ProVate, with emphasis on the ability to insert and remove the device (to exclude hand movement limitation), were recorded in an author-compiled questionnaire. Participants' responses were recorded on a scale ranging from 0 to 4, where 0 means no complaints (not having any complaints at all) and 4 means a high level of complaints. Results were then analyzed and plotted on a scale of 0-100, where 0 indicates total dissatisfaction from device usage and 100 means complete satisfaction. McNemar's tests were used to assess the proportion of participants who scored specific questions as having no complaint at all (0), before and while using the device, for each item separately. Safety was assessed by recording the rate and incidence of anticipated AEs, including vaginal wall trauma (e.g., erosions, abrasions, ulcerations), vaginal/urine infections, pain, spotting, discomfort, de novo or worsening urinary incontinence and constipation, rate and incidence of serious AEs, and rate and incidence of all AEs (anticipated and non-anticipated, serious and nonserious, related and unrelated to the study device). AEs were assessed using one of the following methods: daily diary, scheduled meeting with the investigator, nonscheduled call from the subject, and scheduled weekly telephone call to the subject. The full analysis (FA) set included all eligible participants who used at least one device (even if the insertion process was never completed). The FA set served as the principal analysis set for the safety assessment. The PP analysis set included all participants from the FA set who used the study device models for at least 20 days, with no significant protocol deviation. The PP analysis set served as the principal analysis set for the analyses of the primary and secondary endpoints. Statistical analyses were performed using SAS v9.4 (SAS ® , SAS Institute Cary, NC USA) software. Under the assumptions (100% success), the sample size required to test the null hypothesis at a 5% significance level and with 80% power was at least 36 evaluable participants. # Results The participants' mean age was 60.2 ± 10.5 years, and 52.3% were between 61 and 70 years of age; their mean body mass index was 25.48 ± 4.16 kg/m 2 . Of the 151 births reported, 108 were spontaneous vaginal deliveries, 39 were vaginal and required instrumentation, and four required cesarean delivery. The average weight of the newborns was 3662 ± 449 g. Most participants (81.1%) were postmenopausal, with a mean length of amenorrhea of 14.2 years. Moreover, 13 participants used systemic hormone replacement therapy, and 6 used vaginal estrogen cream. Altogether, 94 usage cycles in three clinics were recorded. This research was a longitudinal study conducted over 2 years with three phases (each phase >6 months apart) that tested slightly different applicators of the device . No changes to the actual Pro-Vate ring were made. Phase A included 33 participants (usage cycles), of whom 20 were also in phase B. Additional participants were recruited for phase C, which was completed by 41 participants using the final applicator (of these, 22 participants already used the device at phase A/B). Altogether, 52 symptomatic women completed this study; of these, 24 completed one usage cycle, 14 completed two usage cycles, and 14 completed three usage cycles with 28-45 days of ProVate use in each usage cycle. Participants who used more than one model were reconfirmed for inclusion and exclusion criteria before the next usage cycle, which was thus considered as an additional usage cycle (altogether: 94). Eighty-seven new participants were screened, of whom 18 were initial screen failures. Additionally, during the study, 8/69 could not be fitted with available sizes (e.g., wide introitus or short vagina), 3/69 discontinued participation because of AEs, 2/69 were withdrawn because of their inability to insert the device by themselves (short hands and inability to bend back), 1/69 opted for surgery, and 3/69 were removed due to violations of study procedures. In total, after device sizing and accommodation, during the device usage period only, 992 ProVate devices were used over 3393 usage days in the PP population, with an average of 36.1 ± 5.70 days per subject, and 1592 devices were used over 3558 study days in the FA (safety) group. ## Objective efficacy: reduction of pop stage The study population included participants with multiple prolapse sites (e.g., anterior and apical). While using the The three phases of the study. Altogether, 94 usage cycles in three clinics were recorded over 2 years with three phases (each phase >6 months apart) Phase A included 33 participants (usage cycles) who completed the study PP, of whom 20 also tested ProVate in phase B. Additional participants were recruited for phase C, which was completed PP by 41 participants of which 22 used the device during phases A/B. Altogether, 94 usage cycles were completed by 52 symptomatic women, of whom 24 completed one usage cycle, 14 completed two usage cycles, and 14 completed three usage cycles. Participants who used more than one model were reconfirmed for inclusion and exclusion criteria before the next usage cycle, which was thus considered as an additional usage cycle ProVate device, the POP reduction was not limited to a specific location. Still, upward distension of the vaginal apex resulted in a flattening and decline of the prolapse at other vaginal sites. General efficacy analyses were conducted on the PP set using 94 usage cycles. However, in two cases, the POP-Q results after the study were missing. Hence, the reduction in POP was calculated over only 92 cases. In the PP set, the pre-study POP-Q staging included 28 usage cycles (29.79%) with stage 2 prolapse and 66 (70.21%) usage cycles with stage 3 prolapse [fig_ref] Figure 4: Comparison of objective efficacy [/fig_ref] , all symptomatic. In all usage cycles (100%), a reduction of at least two POP-Q stages while using the Pro-Vate device (95% exact confidence interval [CI] [96.07; 100]) was observed [fig_ref] Table 2: Prolapse reduction while using the ProVate Device [/fig_ref]. Besides, in 64 of 66 (97%) cases with POP-Q stage 3 prolapse, a reduction of three POP-Q stages (95% exact CI [89.48; 99.63]) was observed. Therefore, it is evident that the first objective efficacy endpoint was met, and the null hypothesis was rejected (P < 0.001). A secondary objective efficacy endpoint relates to the proportion of participants who eventually had either POP-Q stage 0 or 1 prolapse at the end visit while using ProVate. There was no prolapse (POP-Q stage 0) in 90 usage cycles (97.8%), whereas there was POP-Q stage 1 prolapse in two usage cycles (2.2%; [fig_ref] Table 2: Prolapse reduction while using the ProVate Device [/fig_ref]. Collectively, in 92 of 92 cases (100%), prolapse was reduced to either stage 0 or 1 (P < 0.0001). This improvement was demonstrated at all three study clinics with no statistically significant difference among sites; hence, data from all study sites were pooled. shows results from the author-compiled POP symptoms alleviation scores obtained from visit 1 (before device use) and visit 5 (end visit). The scores were substantially reduced for each of the 10 items, and the mean total score decreased significantly from 29 to 2.7 (P < 0.0001). ## Subjective efficacy: reduction of pop symptoms ## Qol questionnaires A statistically significant decrease in all items (implying an improvement in QoL regarding POP) was observed on both the modified PFDI-20 and PFIQ-7 QoL questionnaires. shows a substantial decrease in all items of the modified PFDI-20 scores. The mean total score decreased from 33.6 before using ProVate to 5.1 while using ProVate (P < 0.0001). The proportion of participants who scored "not at all" for specific items ranged from 5.3 to 76.6% at baseline. Subsequently, this value increased (80.6 to 98.9%) at the study end (P < 0.0001). demonstrates a substantial decrease in all modified PFIQ-7 items scores. The mean total score decreased from 24.9 before using ProVate to 0.7 while using ProVate (P < 0.0001). The proportion of participants who scored "not at all" for specific items ranged from 33.0 to 81.9% at baseline. However, these values increased to 95.7% at the study end (P < 0.0001). ## Subject's satisfaction score As shown in [fig_ref] Figure 8: ProVate Satisfaction Score [/fig_ref] , the responses to the satisfaction questionnaire were highly favorable. This figure shows only the results with scores of "not having any complaints at all" given for all 14 items, ranging from 77.7 to 100%. Most items scored at least 90%. ## Aes General safety analyses were conducted on the FA set; 98.9% of AEs were mild, and 87.9% were anticipated. There were no device-related serious AEs, and all AEs resolved completely with no sequelae. In all three study parts, while 124 AEs were reported in the FA set, 109 AEs were observed in the PP set. The most common AEs were discomfort and spotting, anticipated for all devices used vaginally during initial usage stages. [fig_ref] Table 3: Summary of device-related adverse events [/fig_ref] shows the distribution of device-related AEs within the FA and PP sets. In the FA set, 91 AEs occurred in 51 participants (45.95% of the FA set) while using 1592 devices over 3558 usage days. However, in the PP set, 77 AEs occurred in 44 participants (46.81% of the PP set) while using 992 devices over 3393 usage days. Seven cases of vaginal wall trauma (accounting for 7.6% of device-related AEs) were seen at the beginning of the study in only 4 (3.6%, FA set) participants. ## Fig. 5 Comparison of complaints before using ProVate and while using ProVate. Complaints were graded 0-4 using the POP alleviation score (0 being "no complaint at all" and 4 being "significant complaint"). Scores on visit 1 (before using the device) and visit 5 (while using the device) were normalized to the 0-100 scale and were analyzed and compared (94 usage cycles, 992 devices, 3393 usage days, PP population; mean total score p < 0.0001, POP = Pelvic Organ Prolapse). (Source: ConTIPI Medical Ltd., with permission) Modified PFDI-20 Quality of life questionnaire. Comparison of POP-relevant questions before using ProVate and while using ProVate. Complaints were graded 0-4 (0 being "no complaint at all" and 4 being "significant complaint"). Scores on visit 1 (before using the device) and visit 5 (while using the device) were normalized to the 0-100 scale and were analyzed and compared (94 usage cycles, 992 devices, 3393 usage days, PP population; mean total score p < 0.0001, POP = Pelvic Organ Prolapse, PFDI-20 = Pelvic Floor Disability Index 20) Modified PFIQ-7 Quality of life questionnaire. Comparison of POP-relevant questions before using ProVate and while using ProVate. Responses were graded 0-3 (0 being "no complaint at all" and 3 being "significant complaint"). Scores on visit 1 (before using the device) and visit 5 (while using the device) were normalized to the 0-100 scale and were analyzed and compared (94 usage cycles, 992 devices, 3393 usage days, PP population; mean total score p < 0.0001, POP = Pelvic Organ Prolapse, PFIQ-7 = Pelvic Floor Impact Questionnaire, short form 7) The largest proportion of AEs consisted of sporadic AEs, usually one to two complaints each. In the 91 potentially device-related AEs (FA set), 33 (36.3%) were reported during sizing and 58 (63.7%) during the device usage phase. Most of the device-related AEs occurred within 1 week from the study start (58.9%) and during the use of the first five devices (75.8%). This finding shows a typical learning curve for the new product. ## Specific safety points: vaginal infections, urinary tract infection, and urine retention There were no signs or symptoms of vaginal infections (based on self-report or vaginal examination). However, one case of urinary tract infection (UTI) was observed, which was treated with antibiotics. A case of presumptive UTI was also observed, in which the physician commenced treatments without a lab test and without reporting to the site. PVR urine volume was studied by ultrasound scan before insertion of the first device and with the device deployed within the vagina (visit 4). There was no significant difference in PVR between before (15.0 ± 15.56 mL [range, 0-53.5 mL]) and while using the ProVate (14.1 ± 21.9 mL [range, 0-90.7 mL]). # Discussion While offering new aspects of home self-insertion and removal of a disposable device, this study aimed to demonstrate the substantial objective and subjective efficacy and safety of ProVate, together with the reported increase in QoL and users' satisfaction. The primary objective efficacy endpoint of the study, the proportion of participants with at least a one-stage reduction in the POP-Q stage, was met and shown to be 100%. In >96%, two or more stages on POP-Q were reduced. In >97%, prolapse was reduced to stage 0, regardless of the initial stage. The author-compiled POP symptoms alleviation score showed significant improvement in prolapse symptoms while using the device, reflecting substantial subjective efficacy, regardless of the objective efficacy. The prolapse reduction from using ring pessaries was associated with increased QoL [bib_ref] Cross sectional study on assessment of ring pessary cleaning and removal every..., De Albuquerque Coelho [/bib_ref]. In this study, the participants' QoL improved considerably with ProVate. This finding was reflected in the results from the modified PFIQ-7 and PFDI-20. Furthermore, as indicated by the specific questionnaire, the users' satisfaction was significant. Medical literature cites conflicting data on the prevalence of AEs within groups of pessary users. For example, although Hanson et al. [bib_ref] Vaginal pessaries in managing women with pelvic organ prolapse and urinary incontinence:..., Hanson [/bib_ref] reported that only 14.5% of users had any complaints, Bai et al. [bib_ref] Survey of the characteristics and satisfaction degree of the patients using a..., Bai [/bib_ref] reported that 73.1% experienced AEs, and Sarma et al. [bib_ref] Long-term vaginal ring pessary use: discontinuation rates and adverse events, Sarma [/bib_ref] reported that 56% had AEs. This large variability reflects a difference in reporting. Here an ongoing daily/weekly follow-up of complaints, together with a daily diary, will likely lead to a much larger proportion of complaints. However, the rate of AEs was still relatively low with ProVate, and most AEs were minor, mild, and anticipated. Most anticipated AEs within the study occurred during the sizing phase and at the first week of the usage phase. These issues diminished when participants became more experienced with the use of the device ("learning curve effect"). Discomfort and spotting accompany the initial use of any intravaginal device, including menstrual tampons, mainly in those with estrogen deprivation [bib_ref] Effect of vaginal estrogen on pessary use, Dessie [/bib_ref]. As anticipated, these two were the most prevalent AEs upon the initial usage of ProVate. However, their incidence was low and diminished with usage experience. Vaginal wall trauma, an anticipated AE, occurs in 3-24% of pessary users [bib_ref] Natural history of pessary use in women aged 65-74 versus 75 years..., Ramsay [/bib_ref]. In this study, the low rate of vaginal wall trauma (3.6%) is attributable to the user's inexperience and initial trials of inserting the device. This mechanism of wall trauma is probably different from where trauma is caused by prolonged pressure [bib_ref] Management of the neglected vaginal ring pessary, Fernando [/bib_ref] ("pressure ulcers") by a reusable pessary. Vaginal purulent discharge, itching, and foul smell are common among pessary users [bib_ref] How often should ring pessaries be removed or changed in women with..., Miceli [/bib_ref] [bib_ref] One-year prospective comparison of vaginal pessaries and surgery for pelvic organ prolapse..., Lone [/bib_ref] , specifically among those who use them for long periods, perhaps due to biofilm formation [bib_ref] Garland SM Bacterial biofilm formation on vaginal ring pessaries used for pelvic..., Gould [/bib_ref]. However, no complaints, clinical signs, or symptoms of vaginal infection arose in this study while using ProVate. This finding is attributed to the frequent replacement of a fresh and clean device. Compared with most other studies that rely on memory recall only, a strength of this study is the daily collection of AEs in a diary of more than 3558 usage days. Additional strengths include the long usage period (3558 usage days) under strict supervision and the design of the study whereby a strict patient follow-up was used, which allowed for the early detection of specific AEs (e.g., vaginal wall trauma) and corrective actions (e.g., instructing participants how to properly insert the device). Another strength of the study is its longitudinal design conducted over 2 years, enabling adequate follow-up on possible late AEs and possible prolonged usage habits. A limitation of this study is its single-arm design, with data collected during use of the ProVate device only. However, it should be noted that established and validated key performance indicators (POP-Q, PFDI-20, PFIQ-7) were used for the main comparisons to investigate functionality, performance, and usability, and that a comparison with existing ring pessaries was not necessary to establish required data and thus was not included in the current study. Therefore, a future study comparing results between ProVate and existing ring pessaries is needed. Another limitation is the rather short follow-up duration of up to 45 usage days within each usage cycle; however, this is undoubtedly sufficient to demonstrate the efficacy and safety of a new device. Simultaneously, participants who repeated usage within up to three usage cycles in 2 years actually demonstrated prolonged usage habits. # Conclusion In an era where many women avoid clinic visits (e.g., during a pandemic) and when avoidance of treatment can lead to complications, ProVate is an example of a simple and available self-use home management method. This new management modality for POP may elevate compliance with POP treatment among untreated patients. However, further studies are needed to learn more about other characteristics of this device. [fig] Figure 4: Comparison of objective efficacy (POP-Q staging) before and while using the ProVate Device. Before using ProVate, 70.21% of users had POP-Q stage 3 prolapse, whereas 29.79% had stage 2 prolapse. While using ProVate, 100% of subjects had a substantial reduction of prolapse to POP-Q stage 0/1. (POP-Q = Pelvic Organ Prolapse Quantification) [/fig] [fig] Figure 8: ProVate Satisfaction Score. Chart shows only responses of "no complaints at all" by study end. Responses were graded 0-4 (0 being "no complaint at all" and 4 being "significant complaint"), normalized to the 0-100 scale, analyzed, and compared (94 usage cycles, 992 devices, 3393 usage days, PP population) [/fig] [table] Table 1: Identified downsides of existing ring pessaries and possible mitigationsIdentified problems with currently available ring pessaries which cause women to refrain from using them or to discontinue usage, as well as possible mitigation options [/table] [table] Table 2: Prolapse reduction while using the ProVate Device (POP-Q staging, 94 usage cycles, PP set, POP = Pelvic Organ Prolapse, POP-Q = Pelvic Organ Prolapse Quantification) a Compared with those who had POP-Q stage 3 prolapse POP Reduction Results With The ProVate Device (by POP-Q staging) Results [/table] [table] Table 3: Summary of device-related adverse events (AEs) while using ProVateIn the Full Analysis Set (FAS), there were 111 confirmed/reconfirmed participants in the 3 phases of the study, some participated in more than one phase, >6 months apart. 51 (45.95%) had 1 or more device-related AEs. Altogether there were 91 device-related adverse events while using 1592 devices over 3558 usage days In the per-protocol (PP) set, there were 94 completed usage cycles, where in 44 (46.81%) there were 1 or more device-related AEs. Altogether there were 77 devicerelated adverse events while using 992 devices over 3393 usage days SUI Stress Urinary Incontinence, UTI Urinary Tract Infection [/table]

Aconite poisoning with arrhythmia and shock ## 1. Case report A 55-year-male presented in emergency department with history of epigastric burning, nausea, vomiting, dizziness, generalized paresthesia, palpitation, and profuse sweating of 2 h duration. Patient had no history of chest pain. Patient had past history of ischemic stroke 1-year back and residual left hemiparesis. After reading a book of traditional/Ayurvedic medicine, patient decided to self-medicate with ''Bachnaag'' (Aconite) root for residual hemiparesis. He procured Aconite root from Ayurvedic pharmacy and took about five gram of dried root of aconite plant. Patient became symptomatic within 1 h of taking aconite root. On examination, patient was anxious, sweating profusely and had pulse rate of 110/min, irregularly irregular, blood pressure of 80/50 mm of mercury, and respiratory rate of 26/ min. Cardiovascular examination was normal, except for frequent ectopics and ventricular tachycardia. Neurological examination showed a conscious, coherent but anxious patient with residual left hemiparesis. Other systems were normal. Electrocardiogram (ECG) obtained showed multifocal ventricular ectopics with bizarre complexes. Complete blood count, renal functions, and liver profile were normal. Serum electrolytes (sodium: 136 mEq/l, potassium: 3.8 mEq/l and magnesium: 1.8 mEq/l) and cardiac enzymes (creatinine phospho kinase-MB and troponin I) were normal. Patient i n d i a n h e a r t j o u r n a l 6 8 ( 2 0 1 6 ) s 2 0 7 -s 2 0 9 a r t i c l e i n f o Examination revealed shock with irregular pulses. Initial ECG showed frequent multifocal ventricular ectopics (VE), which later turned to short runs of ventricular tachycardia (VT). Immediate gastric lavage was done and activated charcoal given. Patient was treated with fluid resuscitation without any improvement in blood pressure. Patient was started on noradrenaline infusion with gradual recovery from hypotension over a period of 6 h, but support was continued for 48 h. Amiodarone was started to control ventricular excitability, which persisted over 72 h with gradual decrease in frequency of VT and VE. Patient was discharged with normal sinus rhythm on oral amiodarone on 5th day of hospitalization. On follow-up after 2 weeks patient was totally asymptomatic and amiodarone was stopped. was advised coronary angiography but he refused. Arterial blood gases (ABG) parameters were normal. Immediate gastric lavage was done and activated charcoal given. Patient was treated with fluid resuscitation to maintain central venous pressure of 14 cm of H2O but there was no improvement in blood pressure. Patient was started on noradrenaline infusion. Blood pressure improved gradually over a period of 6 h but patient required inotropic support for next 48 h. As there was persistent multifocal ventricular activity, patient was also started on amiodarone infusion. Ventricular excitability persisted over 72 h with gradual decrease in frequency of ectopics. Patient was discharged in normal sinus rhythm on oral amiodarone on 5th day of hospitalization. On follow-up after 2 weeks patient was totally asymptomatic and amiodarone was stopped. # Discussion Aconitum also known as aconite, monkshood, women's bane, devil's helmet, Queen of all Poisons, or blue rocket is a genus of over 250 species of flowering plants belonging to the family Ranunculaceae 1,2 . In India, it is also called as Bachhnaag, Meetha Vish, Vatsanagaka, Vatsanabha or Visa vajranaga. Different species of Aconitum have been used for centuries both as poisons and medicines. It is still being used in traditional medicines of India, China, and Japan. The toxicity of the extracts follows the same order as the alkaloid content: roots, flowers, leaves, and stems. [bib_ref] Clinical features and management of herb-induced aconitine poisoning, Lin [/bib_ref] Aconite is a fast-acting toxin. The active principles are aconitine and related alkaloids. Pure aconite 2 mg or aconite plant 1 g may cause death. [bib_ref] Aconite poisoning, Singh [/bib_ref] Aconitine's toxicity is characterized by a burning sensation of the lips, tongue, mouth, and throat almost immediately following ingestion; it may be followed by numbness of the oral cavity and throat and difficulty in speech. Salivation, nausea, vomiting, dizziness, syncope, palpitation, and diarrhea may occur. There may be visual blurring or yellowgreen color vision distortion, weakness, and incoordination. Toxicity mainly affects cardiovascular system and neuromuscular system. Refractory ventricular arrhythmias and refractory hypotension may lead to death. [bib_ref] Clinical features and management of herb-induced aconitine poisoning, Lin [/bib_ref] [bib_ref] Aconite poisoning, Singh [/bib_ref] [bib_ref] Aconite poisoning, Chan [/bib_ref] The cardiotoxicity and neurotoxicity of aconitine and related alkaloids are due to their actions on the voltage-sensitive sodium channels of the cell membranes of excitable tissues, including the myocardium, nerves, and muscles. Aconitine binds with the voltage-sensitive sodium channels thereby causing a persistent activation of the sodium channels, which become refractory to excitation. Arrhythmia is triggered due to delayed after-depolarization and early after-depolarization. The arrhythmogenic properties of aconitine are also in part due to its anticholinergic effects mediated by the vagus nerve. It has hypotensive and bradycardic actions due to activation of the ventromedial nucleus of the hypothalamus. Through its action on voltage-sensitive sodium channels in the axons, aconitine [ ( F i g . _ 1 ) T D $ F I G ] i n d i a n h e a r t j o u r n a l 6 8 ( 2 0 1 6 ) s 2 0 7 -s 2 0 9 blocks neuromuscular transmission by decreasing the evoked quantal release of acetylcholine. [bib_ref] Aconite poisoning, Chan [/bib_ref] Accidental toxicity and death have resulted when the plant has been consumed accidentally, possibly mistaken for wild parsley, horseradish, or other herbs growing in the wild. [bib_ref] A case of fatal aconitine poisoning by monkshood ingestion, Pullela [/bib_ref] [bib_ref] Cardiotoxicity after accidental herb-induced aconite poisoning, Tai [/bib_ref] Few reports of toxicity from western countries have been related to the use of traditional Chinese remedies sometimes with fatal outcome. [bib_ref] Bidirectional ventricular tachycardia resulting from herbal aconite poisoning, Smith [/bib_ref] [bib_ref] Malignant ventricular arrhythmias due to Aconitum napellus seeds, Imazio [/bib_ref] Most incidents of aconite toxicity have been reported from Asian countries with few case reports from India. [bib_ref] Clinical features and management of herb-induced aconitine poisoning, Lin [/bib_ref] [bib_ref] Aconite poisoning, Singh [/bib_ref] [bib_ref] Aconite poisoning, Chan [/bib_ref] [bib_ref] Cardiotoxicity after accidental herb-induced aconite poisoning, Tai [/bib_ref] [bib_ref] reversible panconduction defect and syncope following selfmedication with a homeopathic medicine, Guha [/bib_ref] [bib_ref] Sustained ventricular tachycardia in a case of aconite poisoning, Gupta [/bib_ref] [bib_ref] Successful treatment of aconitine induced life threatening ventricular tachyarrhythmia with amiodarone, Yeih [/bib_ref] Cardiac arrhythmias arising from aconite poisoning has been successfully treated with amiodarone 11 and in resistant cases with charcoal hemoperfusion. [bib_ref] Acute aconitine poisoned patients with ventricular arrhythmias successfully reversed by charcoal hemoperfusion, Lin [/bib_ref] Hypotension resistant to inotropes like dopamine and nor-adrenaline has been successfully treated with ventricular-assist device. [bib_ref] Aconite poisoning managed with a ventricular assist device, Fitzpatrick [/bib_ref] A case series published by Dwivwdi et al. with twelve patients highlights that cardiotoxic effects of ''safe'' herbs/ minerals preparation are notuncommon. 14 Other cardiotoxic herbs include well-known herbs like Digitalis, Areca catechu, and Belladonna and lesser known herbs like Thevetia peruviana, Cleistanus collinus, Mandragora officinarum, Ephedra distachya, and licorice. [fig] Figure 1 -: (A) ECG at the presentation. (B) ECG after 24 h. [/fig]

Mast Cell Sarcoma of the Retroperitoneum With Concurrent Systemic Mastocytosis and an Undisclosed Associated Hematologic Neoplasm: A Case Report # Introduction Mastocytosis arises from a clonal, neoplastic proliferation of mast cells that accumulate in one or more organ systems.Mast cell sarcoma (MCS) is an exceedingly rare variant of mastocytosis identified by morphologically atypical mast cells that are locally destructive and highly aggressive. [bib_ref] Mast cell sarcoma: new cases and literature review, Monnier [/bib_ref] We hereby present a unique case of retroperitoneal MCS with concurrent systemic mastocytosis. An associated hematological neoplasm, suspected to be a clonal myeloid disorder, is also noted. ## Case presentation A 23-year-old adult was a case of 46, XY disorder of sex development (46 XY DSD) (so-called male pseudohermaphrodite). Before DSD was diagnosed by karyotype analysis in May 2020, she had suffered from intermittent facial flushing and palpitation associated with tachypnea and near syncope for 2 months. Hormone replacement therapy was tried but without improvement. Four months later, abdominal magnetic resonance imaging (MRI) revealed bulky retroperitoneal and retrocrural lymphadenopathies when she had intermittent abdominal pain and body weight loss of 5 kg for 2 weeks, in addition to persistent intermittent flushing and palpitation. Subsequent pathology interpretation of computed tomography (CT)-guided biopsy of retroperitoneum mass was spermatocytic seminoma according to morphologic pictures [fig_ref] Figure 1: The morphology of the specimen from biopsy of retroperineum mass shows a... [/fig_ref] and the expression of CD30 and CD117 immunohistochemically. The SRY mutation (c.60delT), responsible for DSD, was confirmed soon thereafter. For the treatment of spermatocytic seminoma, the patient underwent one course of chemotherapy with bleomycin, etoposide, and cisplatin. After chemotherapy, the patient had temporary relief of abdominal pain, but symptoms recurred soon after. One episode of flushing and palpitation, followed by shock with conscious change and seizure, occurred during hospitalization. The patient recovered quickly and no specific etiology could be identified. At the same time, we noticed that regular antihistamines and antipyretics as needed could relieve her palpitation and flushing. Considering all these atypical presentations, open biopsy was suggested. A few days later, oliguria was noted, along with elevated creatinine (2.42 mg/dL), uric acid (11.2 mg/dL), and LDH (1383 U/L). Incisional biopsy over the right pelvic lymph node (LN), right ovary, and retroperitoneal tumor and bilateral double-J catheter placement for obstructive nephropathy caused by tumor compression was performed. Her renal function improved temporarily after the surgery; however, only days after the operation, oliguria, dyspnea, pulmonary edema, and pleural effusion were found. An abdominal CT scan found multiple new liver metastases, and progressive retroperitoneal tumors with necrosis. Shock and consciousness change developed suddenly 2 days later. No evidence of brain metastases but a 2.6 × 0.5 cm lesion over the left occipital scalp with bony erosion was revealed on brain CT scan. ## 2 ## Clinical pathology Mastocytosis in ovarian tissue was reported first [fig_ref] Figure 2: Morphologically, the tumor cells of ovary show diffuse infiltration of mast cells... [/fig_ref] and b). Meanwhile, bone marrow aspiration was suggested for progressive unexplainable leukocytosis during this month (white cell count 34 700/μL with neutrophils 84.8%, lymphocytes 5.6%, monocytes 1.5%, eosinophils 2%, and metamyelocytes 3%, hemoglobin 8.9 g/dL and platelets 169 000/µL on the day of conscious change). However, her family refused. Her condition went downhill rapidly, and the distributive shock became refractory 2 days after conscious change (1 week after biopsy). The patient expired on the following day. The final pathological reports of the retroperitoneal tumor and LN were confirmed shortly after the patient's death. Morphologically, there were diffuse infiltrations of atypical round-shaped epithelioid tumor cells, with the majority showing prominent nucleoli and eosinophilic to amphophilic cytoplasm, while scattered cells had hyperchromatic nuclei without nucleoli located peripherally resembling mast cells [fig_ref] Figure 2: Morphologically, the tumor cells of ovary show diffuse infiltration of mast cells... [/fig_ref]. Conspicuous tumor necrosis s also noted in the retroperitoneal specimen. Immunohistochemistry staining (IHC) showed diffuse positivity for CD117, CD30, CD33, CD43, tryptase and focal positive for CD25, CD68 and negative for CD2, myeloperoxidase and lysozyme [fig_ref] Figure 3: Immunohistochemically, the tumor cells of retroperitoneal specimens show strong positivity for CD30 [/fig_ref]. The neoplastic cells in LN were also diffusely positive for CD117 and tryptase immunostaining. Therefore, the diagnosis of SM with retroperitoneal MCS was impressed. In addition, the C-kit mutation D816V was demonstrated in retroperitoneal tumor, pelvic LN, and ovary. The serum tryptase level before death was >200 μg/lL. Karyotype of peripheral blood checked on her final day revealed 46 to 54, XY, −1, +2, +rea(4q), +5, +6, rea(12q), rea(13p), −14, add(14)(p10), add(15)(p10), i(17) # Discussion Although MCS was described as early as 1986 by Horny et al, [bib_ref] Mast cell sarcoma of the larynx, Horny [/bib_ref] the diagnosis of MCS remains challenging. The morphology of MCS is highly heterogeneous and may differ from site to site within the same patient. [bib_ref] Mast cell sarcoma: new cases and literature review, Monnier [/bib_ref] Likewise, IHC in MCS is complicated. In MCS, highly atypical mast cells usually express tryptase, CD117, and CD68 and are negative for CD25 in 25% of the cases and negative for CD2 in 48% of the cases. 2 CD30, which has been suggested as a marker for SM aggressiveness, is expressed in 42% of tested MCS samples. [bib_ref] Mast cell sarcoma: new cases and literature review, Monnier [/bib_ref] In our case, the initial pathological diagnosis of a retroperitoneal mass was misled as spermatocytic seminoma because of the history of DSD, location of the lesion and indistinctive morphological and IHC features. The clue leading us to the suspicion of MCS for retroperitoneal mass was diffuse mast cell infiltration in the ovary sample. Notably, despite the high sensitivity and specificity of tryptase in MCS, it is possibly only stained under a special stain protocol. [bib_ref] Aggressive systemic mastocytosis with sarcoma-like growth in the skeleton, leukemic progression, and..., Krauth [/bib_ref] Similarly, tryptase staining in our case was much stronger after recombining the reagents. Therefore, the presence of atypical cells positive for CD117 and/or tryptase could lead to the proper suspicion of MCS, which is the major step of correct diagnosis. [bib_ref] Mast cell sarcoma: new cases and literature review, Monnier [/bib_ref] The diffused infiltration of spindle-shaped mast cells in ovary not only guided us to the diagnosis of MCS, but also fulfilled 1 major and 1 minor criterion of SM. The spectrum of systemic mastocytosis was broad. According to the WHO diagnostic criteria for SM, our case fulfilled 1 major SM criterion and 3 minor criteria, including spindle-shaped mast cells in extracutaneous tissues, mutational analysis of KIT showing codon 816 mutation, and extracutaneous mast cells expressing CD25. Thus, the diagnosis of SM was confirmed, but could not be classified as aggressive SM because concurrent MCS masked the possible presentation of aggressive SM. In addition, the progressive and unreasonable leukocytosis (neutrophilia 29 426/μL but not mast cells in peripheral blood) represents the existence of bone marrow disorder per se. Due to the patient's refusal of bone marrow examination, the exact diagnosis of bone marrow disorder could not be obtained, nor could aleukemic mast cell leukemia or bone marrow involvement of MCS or SM be excluded. [bib_ref] Aleukemic mast cell leukemia (AMCL) -clinical characteristics and outcomes, Jain [/bib_ref] Nevertheless, much evidence can be drawn from the karyotype of peripheral leukocyte, which is indicative of chronic myeloid leukemia (the presence of i(17)), myeloproliferative disorder (the presence of i(17) and del(20)), myelodysplastic syndrome (the presence of i(17), del(20) and +19), or even acute myeloid leukemia (the presence of i(17), del(20) and +19). [bib_ref] Hematologic malignancies, critical genes and representative pictures for 166 chromosome anomalies, Mahaffey [/bib_ref] Taken together with extreme neutrophilia, a clonal myeloid neoplasm is postulated. Taking into account of the concurrent diagnosis of SM and suspected clonal myeloid neoplasm, the final diagnosis of SM-AHN was made. This case demonstrates the features of both SM and MCS. Of the 26 cases of MCS published in the literature, only 4 had a history of associated SM. [bib_ref] Aggressive systemic mastocytosis with sarcoma-like growth in the skeleton, leukemic progression, and..., Krauth [/bib_ref] [bib_ref] Mast cell sarcoma of the scalp: the first sign of undisclosed systemic..., Falleti [/bib_ref] [bib_ref] Mast cell sarcoma mimicking metastatic colon carcinoma, Schwaab [/bib_ref] The first was a case of aggressive SM with sarcoma-like growth of mast cells in the skeleton, and progression to mast cell leukemia. [bib_ref] Aggressive systemic mastocytosis with sarcoma-like growth in the skeleton, leukemic progression, and..., Krauth [/bib_ref] The second was a case of MCS of the scalp, which appeared to be the first ## 4 Clinical Pathology manifestation of underlying SM. [bib_ref] Mast cell sarcoma of the scalp: the first sign of undisclosed systemic..., Falleti [/bib_ref] The third was a case of aggressive SM associated with chronic eosinophilic leukemia with transformation into a "secondary" bifocal MCS over the colon and liver. [bib_ref] Mast cell sarcoma mimicking metastatic colon carcinoma, Schwaab [/bib_ref] Last, there was a case of bone marrow mastocytosis with an associated secondary extramedullary MCS over the retroperitoneum, testis, and heart, proven by autopsy.Therefore, just as how MCS could lead a progressive course to transform into an aggressive generalized disease; it could also be an initial presentation of a systemic disease. In addition, while somatic KIT mutations are detected in 90% of SM, 10 50% of cases of MCS have wild-type KIT. [bib_ref] Mast cell sarcoma: new cases and literature review, Monnier [/bib_ref] The most common KIT mutation is D816V, which was also detected in neoplastic mast cells from the ovary, pelvic LN, and retroperitoneal mass in our patient. Taken together, it is proposed that our patient had SM with secondary MCS. A subcategory of secondary MCS is not included in the WHO classification, 1 but the term "secondary" MCS has been used to describe the case of SM with transformation to MCS. [bib_ref] Aggressive systemic mastocytosis with sarcoma-like growth in the skeleton, leukemic progression, and..., Krauth [/bib_ref] Our case developed a clonal myeloid neoplasm in a short time after the clinical onset of MCS. Although the KIT D816V mutation could be detected in clonal hematological non-mast cells in SM-AHN (30% and 20% cases of SM-acute myeloid leukemia (SM-AML) and SM-MPN respectively), [bib_ref] Variable presence of KITD816Vin clonal haematological non-mast cell lineage diseases associated with..., Sotlar [/bib_ref] the KIT status of blood cells in our case was not checked. An abnormal karyotype, found in 26% of cases with SM-AHN but uncommon in SM, [bib_ref] Cytogenetic abnormalities in systemic mastocytosis: Who subcategory-specific incidence and prognostic impact among..., Shah [/bib_ref] corresponds to our situation. Therefore, the mechanism of SM, MCS and a non-MC hematological neoplasm transformation is unclear, but studies support a multi-hit theory in developing SM-AHN, and KIT mutation might occur before, or more often, after the non-KIT mutation. [bib_ref] Mastocytosis: 2016 updated who classification and novel emerging treatment concepts, Valent [/bib_ref] Due to the complexity of the mechanism, multiple organ involvement, and convoluting clinical course of mast cell disorders, collaborative multidisciplinary patient care and pooling of experience and knowledge from different specialty is crucial for making the correct diagnosis and arranging optimal and timely treatment. [bib_ref] Evaluation and classification of mast cell disorders: a difficult to manage pathology..., Leru [/bib_ref] In summary, we present a unique case of MCS with concurrent systemic mastocytosis and an undisclosed associated hematological neoplasm (SM-undisclosed AHN), with a rapidly progressing disease burden eventually leading to mortality. Despite unknown bone marrow status at disease burden, it is worth noting that our case draws attention to a subvariety of SMs that may progress to MCS, rapidly accompanied by the existence of an associate hematological neoplasm. [fig] Figure 1: The morphology of the specimen from biopsy of retroperineum mass shows a picture of proliferation of neoplastic cells in sheet pattern with adjacent loose stromal tissue having inflammatory cells infiltration, including lymphocytes and eosinophils (a and b). [/fig] [fig] Figure 2: Morphologically, the tumor cells of ovary show diffuse infiltration of mast cells with spindle shapes (a and b). The tumor cells of retroperitoneum show diffuse infiltration of atypical round-shaped epithelioid tumor cells, with the majority showing prominent nucleoli and eosinophilic to amphophilic cytoplasm, while scattered cells have hyperchromatic nuclei without nucleoli located peripherally resembling mast cells (c and d). [/fig] [fig] Figure 3: Immunohistochemically, the tumor cells of retroperitoneal specimens show strong positivity for CD30 (a), CD117 (b), and strong and diffuse positivity for mast cell tryptase (c). The tumor cells of ovarian specimens show diffuse expression of mast cell tryptase (d). [/fig]

The effects of anticholinergic medications on cognition in children: a systematic review and meta-analysis Cognitive side effects of anticholinergic medications in older adults are well documented. Whether these poor cognitive outcomes are observed in children has not been systematically investigated. We aimed to conduct a systematic review and meta-analysis on the associations between anticholinergic medication use and cognitive performance in children. Systematic review was conducted using Medline, PsychInfo, and Embase, identifying studies testing cognitive performance relative to the presence versus absence of anticholinergic medication(s) in children. We assessed effects overall, as well as relative to drug class, potency (low and high), cognitive domain, and duration of administration. The systematic search identified 46 articles suitable for meta-analysis. For the most part, random effects meta-analyses did not identify statistically significant associations between anticholinergic exposure and cognitive performance in children; the one exception was a small effect of anticholinergic anti-depressants being associated with better cognitive function (Hedges' g = 0.24, 95% CI 0.06-0.42, p = 0.01). Anticholinergic medications do not appear to be associated with poor cognitive outcomes in children, as they do in older adults. The discrepancy in findings with older adults may be due to shorter durations of exposure in children, differences in study design (predominantly experimental studies in children rather than predominantly epidemiological in older adults), biological ageing (e.g. blood brain barrier integrity), along with less residual confounding due to minimal polypharmacy and comorbidity in children.OPEN Anticholinergic medications are commonly prescribed 1-3 yet a growing body of evidence has demonstrated that their use is associated with a higher risk of incident cognitive impairment. This literature has been reviewed multiple times in older adults, whereby anticholinergic medications have been consistently associated with cognitive decline and dementia. There has been no systematic synthesis of the cognitive effects of anticholinergic medications in children. There are few population-based studies that have assessed the extent to which children are exposed to anticholinergic medicines. Most studies examining anticholinergic medicines in children have focussed on the use of medicine classes for specific indications, for example, asthma or overactive bladder, rather than providing population-based estimates for the use of anticholinergic medicines like the studies in older adults. Approximately 11% of Australian children have a current diagnosis of asthmaand up to 20% of children experience bedwetting 11 so there is potential for a high prevalence of use of anticholinergic medicines to treat these conditions in children. One population based study from Slovenia reported that 20% of children using prescription medicines were dispensed anticholinergic medicines, most commonly antihistamines. Anticholinergic medications refer to a broad class of medicines which block the neurotransmitter acetylcholine. These medications are used in the treatment of many conditions such as depression, vertigo, asthma, cardiac arrhythmias and incontinence. High potency anticholinergic medications appear to most detrimentally affect cognition in older adults (as compared to low potency). Further, the class of anticholinergic medication differentially associates with cognitive decline in late-life, with anti-depressants (amitriptyline, www.nature.com/scientificreports/ dosulepin, paroxetine), urologicals (oxybutynin, tolterodine), and antiparkinsonian drugs showing the strongest associations with incident dementia. Neurobiologically, the cholinergic system primarily mediates attentional processesand therefore could be expected to be primarily impaired by anticholinergic medications, although cognitive domain specific effects have not been investigated. The current study aims to quantitively synthesise the literature on associations between anticholinergic medications and cognitive performance in children. Findings from this review will inform medical practitioners of any risks (or lack thereof) associated with anticholinergic use in children, and subsequently help to inform the safe prescribing of anticholinergics. It is critical to identify whether anticholinergics should be prescribed with restraint in children. We hypothesise that in children (1) exposure to anticholinergic medications will be significantly negatively associated with performance on cognitive tests, and that associations will be strongest for (2) antidepressant and urological drug classes (as compared to other drug classes), (3) high-potency anticholinergics (as compared to low-potency), (4) those exposed long-term (as opposed to short-term) and (5), within the cognitive domain of attention. # Methods Search strategy. This study adhered to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines (seefor PRISMA Checklist). A systematic literature search was conducted in December 2019 using the electronic databases Medline, PsychInfo, and Embase. The search strategy used a combination of keywords for anticholinergic medications (see Supplementary Material), cognition terms (cognit* OR neuropsych* OR learn* OR memory OR "executive function" OR "executive functions") and demographic terms (children OR childhood OR youth* OR teen*). No published review protocol exists for the current study. Anticholinergic medications were defined as medicines with clinically significant anticholinergic properties as listed in a systematic review by Duran et al.. Medications assessed by the Duran systematic review to be of either high or low anticholinergic potency, but not ambiguous potency, were included. Studies were screened and assessed for eligibility by two independent reviewers, first by title and abstract, then by full text, according to inclusion and exclusion criteria described below (MC, TJR, DC, CS and JNH). Any conflicts were resolved through consensus. Inclusion and exclusion criteria. Studies of either within-or between-groups design were included if they reported at least one cognitive outcome for both children exposed and unexposed to anticholinergic medications; reported data for a sample of children (< 18 years old); were published in English; and were published in peer-reviewed journal articles. Studies from all publication years were accepted. "On" medication participants included children exposed to at least one anticholinergic medication. "Off " medication participants included matched controls unexposed to any other medication, participants treated with placebo, participants undergoing withdrawal from the medication, or the baseline measurements of the exposed group. To be eligible for inclusion, studies needed to report cognitive outcomes based on objective cognitive measures; subjective behavioural reports were not included (e.g. self, parent or teacher reports of cognitive functioning). Studies were excluded if the control group did not share the same disorder or symptom (i.e. healthy control group) of the experimental group. Studies which only compared the effects of anticholinergic medication versus non-anticholinergic medication, rather than anticholinergic medication versus no medication, were excluded. Studies were also excluded if they involved non-human (animal) participants; if they assessed in-utero anticholinergic exposure; or if they were a case report, case series, thesis or conference abstract. Data extraction. Data were extracted from eligible studies independently by one reviewer (EG, MC, TJR) and then checked by a second reviewer, with any discrepancies resolved through discussion or checked again (by a third reviewer). Extracted data include country of publication, study design, sample size (and number of male/ female participants), age, diagnoses of sample, name of medication, duration of administration, and cognitive domains assessed. The extracted medication name was then classified by potency and drug class by an academic pharmacist (LE). Data required for meta-analysis were also extracted. This included any data for which an effect size (standardised mean difference) could be calculated for differences between on and off medication groups (e.g., means and standard deviations, Cohen's d and confidence intervals (CIs), sample size and correlation statistic, means and correlation statistic, or means and p-value). Quality assessment. A quality assessment tool was developed for this study, adapted from a critical appraisal tool for randomised controlled trials from the Joanna Briggs Institute 20 , see Supplementary Material-Quality Assessment Tool. The Joanna Briggs Institute is a highly regarded organization with recommended 21 and well-used critical appraisal checklists. The quality assessment tool comprised an eight-point checklist. All studies were screened using this tool by two independent reviewers (MC and TJR) and any conflicts in scoring were resolved through discussion. Statistical approach. Some included studies reported data for both within-and between-groups designs. For example, they may include two groups: one that experiences a period of on and off medication, and one non-medicated control group. In these cases, the between-group design (i.e. medication versus control) was preferentially selected in order to minimise the effect of cognitive development (over time). Where one study reported both within-and between-group comparisons for two distinct participant samples (i.e. one group both on and off medication, along with a second group on medication and a third no-medication control group) both within-and between-groups data were extracted. In cases where one study reported both (within and between) www.nature.com/scientificreports/ comparisons over multiple time-points, within-groups data were extracted for any time-points where betweengroups data were unavailable. All outcome measures were standardised using Hedges' g for difference between on-and off-medication groups. A positive Hedges' g represents a better cognitive score for the on-medication group compared to the off-medication group, regardless of the direction of the original cognitive test. Small, medium, and large effect sizes were classified using the Hedges and Olkin 25 method, as 0.20, 0.50, and 0.80 respectively. Comprehensive Meta-Analysis software (version 3) was used to calculate effect sizes, where calculations of Hedge's g are dependent on study design (within-or between-groups). Statistical analyses were conducted using the meta packagefor R (Version 4.0.2). Dependency was present in analyses due to included studies reporting multiple cognitive outcomes or time-points for follow-up based on the same, or largely overlapping, participant samples. This was accounted for by averaging across effect sizes within studies, so one effect size was used per study within each analysis. The data and script associated with this analysis are publicly available (https ://githu b.com/erica ghezz i/antic holin ergic _med_metaa nalys is). Outcomes across studies were pooled using a random-effects model. The commonly used DerSimonian and Laird 27 estimator of between-study variance has been criticised due to its propensity to underestimate true between-study variance, leading to narrow CIs and potential false-positive estimations. Hence, we followed the recommendation of Veroniki et al.and employed the Paule and Mandel 31 method, which has been shown to be less biasedwhen estimating between-study variance. Sensitivity analyses revealed no substantial differences in outcomes when analyses were run using common between-groups estimators. The Hartung-Knapp method for random effects meta-analysiswas also applied to all analyses. A result was considered statistically significant when p < 0.05. We considered this an exploratory study and did not correct for multiple comparisons. Between-study variance was quantified using τ 2 . The proportion of between-study heterogeneity out of total variance was assessed using the I 2 statistic. Values of I 2 were classified as low (25%), moderate (50%), or high (75%). Subgroup analysis. Subgroup analyses were stratified by anticholinergic potency, cognitive domain, drug class, and duration of medication administration. Anticholinergic potency was classified as low or high according to Durán et al.. Cognitive domain was based on Lezak et al.: attention, psychomotor functioning, concept formation and reasoning, perception, memory, executive function, language, and intelligence. The anticholinergic drugs administered were categorised by class as antiepileptics (WHO Anatomical Therapeutic Chemical code N03), antiparkinsonian medicines (N04B), antipsychotics (N05A), antidepressants (N06A), respiratory medicines (R), opioid analgesics (N02A), or urological medicines (G04B). Only one studyreported results based on an antiparkinsonian anticholinergic, so subgroup meta-analysis of this medication class was not conducted (note: the study was included in the overall meta-analysis). Total volume of exposure or dose has been shown to be important in assessing risk of cognitive impairment associated with use of anticholinergic medicines in adults; however, dose was inconsistently reported, or not reported at all, in many of the studies included in the meta-analysis. Duration of exposure, which was consistently reported in the studies, was therefore analysed. Duration of medication administration was categorised as either (1) current and long-term (> 1-month), (2) current and acute (≤ 1-month) and (3) historical administration. Each subgroup analysis was based on a random-effects model, where calculations of within-subgroup variance and comparisons between subgroups were both made using a random-effects model. Fixed effects comparisons of differences between subgroups were not made due to the risk of false positives. The Q statistic was calculated as a test of between subgroups differences. Publication bias. Funnel plots of effect size versus standard error for the primary outcome were visually examined for symmetry to assess for bias across studies due to the small-study effect. As the whole metaanalysis contained at least 10 studies, small-study effect was formally tested using Egger's test of the intercept. If evidence of asymmetry was found (one-tailed p < 0.1 on the Egger's test), Duval and Tweedie's 41 trim and fill method would have been used to quantify the magnitude of potential bias. # Results Summary of studies. A total of 7,645 articles were identified, of which 6,283 were screened by title and abstract following duplicate removal. Full-text review was conducted on 323 articles, and 46 of these were included for final review and meta-analysis. The 46 included studies were published across 6 decades, with 1, 2, 7, 10, 13, and 13 studies published in ascending decades from the 1960s. Of the included studies, 37 were conducted in developed countries, 7 in developing countries, and 2 included children from both developing and developed countries (classified according to the UN 42 ). For a complete overview of the characteristics of included studies, see. Overall cognition. Overall, the 46 studies included reported a total of 536 effect sizes. The pooled effect size of the difference between cognition on and off medication across the 46 studies was negligible and non-significant (g = 0.05, 95% CI − 0.02 to 0.11, p = 0.16; see, with no heterogeneity between studies (τ 2 = 0, I 2 = 0%, Q = 42.36). The funnel plot did not reveal significant asymmetry (Egger's intercept = − 0.5, p = 0.14; see. ## Subgroup analyses. Pooled estimates for subgroup analyses by anticholinergic drug class, potency, length of administration and cognitive domain are presented in. The number of studies within individual subanalyses ranged from 2 to 37. Varying levels of heterogeneity were present across analyses, ranging from null to high (τ 2 range: 0-0.13, I 2 range: 0-76.2, Q = 0. . No significant differences between subgroups were revealed through a test of between-subgroup differences using the random-effects model (see. The pooled effect size for cognitive outcomes on antidepressant medications was small and statistically significant (seeandwww.nature.com/scientificreports/ studies (τ 2 = 0, I 2 = 13.2%, Q = 6.91). Notably, this effect was not significant in a sensitivity analysis (Supplementary which included only studies of high quality. Pooled estimates were non-significant across the remaining anticholinergic drug class (see, potency (see, length of administration (see , and cognitive domain (see subgroup analyses. All null results were replicated within the sensitivity analysis of high-quality studies, except the memory cognitive domain analysis, which had a small positive significant effect (g = 0.09, 95% CI 0.01-0.17, p = 0.02). # Discussion We quantified the effects of anticholinergic medications on cognition in children systematically across the literature. We report that, unlike older adult samples 7-9 , anticholinergic medications are not associated with cognitive impairments in children. This finding was regardless of the classification approach used: drug class, potency, duration of use, and cognitive domain. The discrepancy between child and older adult samples may be due to shorter lengths of exposure in children, higher rates of polypharmacy in older adults, residual confounding, study design, or biological ageing processes. Older adults have the opportunity for years or decades of anticholinergic exposure, with polypharmacy common, whereas studies included here from child samples typically had short exposure durations (6 months or less in most studies) and little polypharmacy. It may be that the detrimental effect of anticholinergic medications on cognition in late-adulthood is driven by long exposure and polypharmacy, factors not observed in children. Further, in late-life, the class of antidepressant appears to differentially affect cognition, with anti-depressants, urologicals, and antiparkinsonian drugs showing the strongest associations with incident dementia risk 4 . We did not see this pattern of effects in children. It may be that duration of exposure and polypharmacy again drives this difference, however residual confounding in late-life samples cannot be ruled out. It may be that incontinence and mood symptoms, for which anticholinergic medications are prescribed, are early clinical indicators of dementiarelated neuropathologies 4 (which accrue decades prior to a dementia diagnosisand that early, undiagnosed dementia is driving the associations between use of anticholinergic medicines and poor cognition in adults. Interestingly, all study designs included in this review were experimental, whereas those included in reviews of older adults are typically longitudinal epidemiological cohort studies 7-9 . Standards of reporting cognitive www.nature.com/scientificreports/ performance also differ between children and adults. Cognitive performance in children is typically reported as test scores on a continuum, while in adults (especially those in late-life), a dichotomous classification of Neurocognitive Disorders is primarily used (e.g. presence versus absence of mild cognitive impairment or dementia). Study designs and differences in classification of cognition therefore may also underlie differences in the patterns of effects observed in children versus older adults, including the finding that anticholinergic antidepressants displayed a positive association with cognition (albeit with a small effect size, which was not significant when only high-quality studies were included). This small positive effect may be due to the short-term nature of the studies included here and is consistent with a meta-analysis of randomised control trials in adult samples. We do not www.nature.com/scientificreports/ www.nature.com/scientificreports/ know the effects of the long-term use anticholinergic antidepressants in children. Notably, a small positive effect of anticholinergic medication on memory was found when only including studies of high quality. Whether this is a true effect, which is counter to that found in adults, needs to be replicated in future studies. Lastly, there are important biological differences between children and adults that would modify the psychopharmacological effects of anticholinergic medications, particularly blood brain permeability. This study is not without limitations. The included studies were biased in terms of geographical representativeness. Fourteen studies were excluded at the full-text stage as they were not in English (of 323) and we do not know if any would have met inclusion criteria; although, given the low number, they would unlikely have changed the conclusions. Authors of papers were contacted, but we either had no response or the author was unable to provide us with the necessary data where it was not presented in text. We assessed the effect of duration of exposure on cognitive outcomes, when total dose or volume of exposure may have been more appropriate. However, this information was inconsistently reported or not reported at all in many of the studies. Therefore, duration of use was used as the best proxy for volume of exposure, with the assumption that longer duration of use would equate to higher volume of exposure. Only 21 of the 100 high-or low-potency anticholinergics identified in a systematic review of anticholinergic medications by Duran et al.were used in the studies included in this meta-analysis. It may be that different results would be seen had children been exposed to a wider range of anticholinergic medicines. Positively, the vast majority of studies (all but two) utilised valid and reliable cognitive outcome measures, as catalogued specifically or adapted from those detailed in Lezak et al.. # Conclusion By pooling effects across previous literature, anticholinergic medications do not appear to detrimentally affect cognitive function in children. In fact, there may be a small positive cognitive benefit of anticholinergic antidepressants, at least in the short-term. Our findings appear to conflict with reviews in older adults, and future studies will have to disentangle the reasons for this. www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ www.nature.com/scientificreports/ ## Data availability All data and code available at https ://githu b.com/erica ghezz i/antic holin ergic _med_metaa nalys is. www.nature.com/scientificreports/

Efficacy and Safety of AbobotulinumtoxinA for the Treatment of Hemiparesis in Adults with Lower Limb Spasticity Previously Treated With Other Botulinum Toxins: A Secondary Analysis of a Randomized Controlled Trial Objective: To examine the safety and efficacy of abobotulinumtoxinA in patients previously treated with botulinum toxin type A (BoNT-A) products other than abobotulinumtoxinA. Design: Secondary analysis from a phase 3, double-blind, single-cycle, randomized, placebo-controlled study. Setting: Fifty-two centers (11 countries). Patients: Adults with spastic hemiparesis were randomized (1:1:1) to receive abobotulinumtoxinA 1000 U, 1500 U, or placebo in their affected lower limb. Main Outcome Measurements: Muscle tone (6-point Modified Ashworth Scale [MAS], 0-5) for the gastrocnemius-soleus complex (GSC); proportion of MAS responders (≥1-point improvement); angle of catch (X V3 ) and spasticity grade (Y) for the GSC and soleus. Assessments were at weeks 1, 4, and 12 post-injection. Only descriptive statistics are presented. Results: Of 388 patients, 84 received previous BoNT-A treatment (abobotulinumtoxinA 1000 U: N = 30; abobotulinumtoxinA 1500 U: N = 28; placebo: N = 26). At week 4, mean (SD) changes in MAS score in the GSC were − 0.8 (1.1), −0.9 (1.0), and − 0.4 (0.7) for abobotulinumtoxinA 1000 U, 1500 U, and placebo, respectively. Greater MAS responder rates were observed for abobotulinumtoxinA versus placebo at all time points. Mean (SD) changes (week 4) for abobotulinumtoxinA 1000 U, 1500 U, and placebo for X V3 were: GSC, 8 (21), 6 (10) and 1 (7); soleus, 11 (21), 5 (9) and 0 (8), respectively; for Y: GSC, −0.4 (0.7), −0.6 (0.8) and − 0.0 (0.9); soleus, −0.5 (0.7), −0.5 (0.7) and − 0.1 (0.6), respectively. Safety data and adverse events were consistent with the overall known profile of abobotulinumtoxinA. Conclusions: Patients previously treated with other BoNT-As showed improved muscle tone and spasticity at week 4 following abobotulinumtoxinA injection versus placebo. These findings suggest that abobotulinumtoxinA, at the recommended doses, has a good safety and efficacy profile in adults with lower limb spasticity who were previously treated with other BoNT-A products. PM R 12 (2020) 853-860 www.pmrjournal.org This is an open access article under the terms of the Creative Commons Attribution License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited. # Introduction Several neurological disorders may give rise to lower limb spasticity (LLS) including stroke and brain/spinal cord injury, cerebral palsy, and multiple sclerosis. [bib_ref] An international survey of patients living with spasticity, Barnes [/bib_ref] AbobotulinumtoxinA (Dysport; Ipsen Pharma, Wrexham, UK), is effective at reducing muscle tone and improving spasticity and functional outcomes in adults with LLS, as shown in several randomized, double-blind studies, [bib_ref] Systematic literature review of abobotulinumtoxinA in clinical trials for lower limb spasticity, Dashtipour [/bib_ref] [bib_ref] Efficacy and safety of abobotulinumtoxinA in spastic lower limb: randomized trial and..., Gracies [/bib_ref] and is licensed in the United States and Europe for the treatment of LLS.In chronic conditions, such as spastic paresis, which require repeat treatment over a prolonged period, immunogenicity (ie, the development of antibodies to the therapeutic product) is a potential concern. [bib_ref] Immunogenicity of botulinum toxins, Naumann [/bib_ref] Although the rate of neutralizing antibodies is low across botulinum toxin type A (BoNT-A) products, secondary nonresponse may occur, which may require the patient to be treated with an alternative BoNT-A product. [bib_ref] Immunogenicity of botulinum toxins, Naumann [/bib_ref] Alternatively, physicians or patients may wish to alter treatment plans based on response to treatment or adverse events, or due to changes in formulary or insurance coverage. [bib_ref] OnabotulinumtoxinA and AbobotulinumtoxinA dose conversion: a systematic literature review, Dashtipour [/bib_ref] However, there are limited data on whether efficacy and dosing requirements of BoNT-A products differ from the label recommendations in patients with LLS who have received previous treatment with other BoNT-A products. A recent phase 3, double-blind, single-cycle study and the open-label multiple-cycle extension demonstrated improved muscle tone after a single abobotulinumtoxinA injection to the affected lower limb in adults with LLS following a stroke or traumatic brain injury. [bib_ref] Efficacy and safety of abobotulinumtoxinA in spastic lower limb: randomized trial and..., Gracies [/bib_ref] Repeated administration was associated with improved walking speed and likelihood of achieving community ambulation, and abobotulinumtoxinA treatment was well tolerated. [bib_ref] Efficacy and safety of abobotulinumtoxinA in spastic lower limb: randomized trial and..., Gracies [/bib_ref] In this double-blind lower limb study, randomization was stratified according to previous treatment status resulting in two subgroups: not previously treated (naïve) patients, who had not received any previous BoNT-A injections to the affected lower limb; and previously treated (non-naïve) patients, who had received at least one previous injection of any BoNT-A product to the affected lower limb. The aim of the present post hoc analyses of the double-blind lower limb study was to assess the safety and efficacy of a single cycle of abobotulinumtoxinA in patients who had been previously treated with any BoNT-A product except for abobotulinumtoxinA. # Methods ## Double-blind lower limb study design The methodology and results of the phase 3, multicenter, double-blind, randomized, placebo-controlled, single-cycle study have been reported previously. [bib_ref] Efficacy and safety of abobotulinumtoxinA in spastic lower limb: randomized trial and..., Gracies [/bib_ref] In brief, patients were randomized 1:1:1 to receive a single injection of abobotulinumtoxinA 1000 U, 1500 U, or placebo. Randomization was stratified according to previous treatment status: not previously treated and previously treated patients, as defined in the preceding text. The total injection volume (7.5 mL) was made up of three vials of either 2.5 mL placebo or reconstituted abobotulinumtoxinA (500 U in 2.5 mL sodium chloride [0.9%]). The content of each 2.5-mL vial was then pulled out into a single 10-mL syringe. Patients randomized to abobotulinumtoxinA 1500 U received three vials of abobotulinumtoxinA, those in the 1000 U treatment arm received two vials of abobotulinumtoxinA and one vial of placebo, and patients in the placebo arm received three vials of placebo. A total volume of 7.5 mL was administered to patients by intramuscular injection including 2.5 mL into the soleus muscle (minimum of three sites), 1.5 mL into the medial and/or lateral gastrocnemius muscle (minimum of two sites), and the remainder of the dose injected into at least one other lower limb muscle selected by the investigator from the following additional distal (tibialis posterior, flexor digitorum longus, flexor digitorum brevis, flexor hallucis longus, flexor hallucis brevis) or proximal (rectus femoris, hamstrings, adductor magnus, gracilis, or gluteus maximus) lower limb muscles. Distal and proximal lower extremity muscles were selected based on their potential role in gait dynamics in paretic patients. [bib_ref] Patient registry of outcomes in spasticity care, Esquenazi [/bib_ref] [bib_ref] Instrumented assessment of muscle overactivity and spasticity with dynamic polyelectromyographic and motion..., Esquenazi [/bib_ref] [bib_ref] Temporospatial parameters of gait after obturator neurolysis in patients with spasticity, Ofluoglu [/bib_ref] Concomitant medications, such as pain medication, anticholinergic drugs, and skeletal muscle relaxants, were kept stable throughout the study. As the protocol defined, the dose, duration and frequency of treatment was left to the clinical discretion of the investigators. Community-based physiotherapy initiated at least 1 month prior to the study entry was continued at the same frequency and intensity until week 4 (or study end if possible). Patients were followed-up for a minimum of 12 weeks and a maximum of 24 weeks. ## Patients Eligible patients were ambulatory adults aged 18 to 80 years with spastic hemiparesis causing gait dysfunction; a comfortable barefoot walking speed of 0.1 to 0.8 m/s, measured on a 10-meter walking test without walking aids; one clinically defined stroke episode or brain trauma ≥6 months before enrollment; toxin-naïve and a Modified Ashworth Scale (MAS) score ≥ 2 in the affected gastrocnemius-soleus complex (GSC) (knee extended), or toxin non-naïve and a MAS score ≥ 3 in the affected GSC (knee extended) at least 4 months after the last injection of BoNT-A in the affected lower limb; and GSC spasticity angle ≥5 (Tardieu scale 11 ; knee extended). Patients were excluded if they had major limitation in passive range of motion at the affected hip, knee, or ankle; a known sensitivity to BoNT-A or abobotulinumtoxinA excipients; severe cognitive impairment that hindered consent provision; or were pregnant. Patients were also excluded if they had received any BoNT-A injections within 4 months of enrollment. ## Post hoc analyses in previously treated patients These post hoc analyses included all patients who had previously been treated with any BoNT-A products except for abobotulinumtoxinA to the affected lower limb at study initiation. Multiple efficacy end points from the double-blind study were included in the present analysis. The primary end point of the double-blind study was change from baseline to week 4 in GSC muscle tone (knee extended), assessed using the MAS (6-point graded scale: 0, 1, 1+, 2, 3, 4; for quantitative analyses, 1+ was considered as 2 and higher numeric scores were incremented to give a MAS range of 0-5). Secondary and tertiary outcome measures assessed in this post hoc analysis were: proportion of MAS responders (≥1 grade improvement from baseline) at weeks 1, 4, and 12; muscle tone (MAS) in the soleus muscle (knee flexed) at baseline, weeks 1, 4, and 12; and spasticity grade in the GSC (knee extended) and soleus muscle (knee flexed) assessed using the Tardieu scale (angle of catch [X V3 ] and severity [Y]) at baseline and weeks 4 and 12. Safety findings, including number of treatment-emergent adverse events (TEAEs), most common TEAEs, and serious adverse events (SAEs) are also reported. # Statistical analysis This was a post hoc analysis and the study was not powered to detect statistical significance. Hence, only descriptive statistics are provided, including mean, standard deviation (SD), and range for continuous variables, and absolute frequency and percentage for categorical variables. Post hoc analyses of efficacy end points were conducted for the intention-to-treat (ITT) population, which included all previously treated patients who received study medication and had data for GSC muscle tone at baseline and week 4. Safety data are reported for the safety population, consisting of all previously treated patients who received study medication. # Results ## Study population A total of 388 patients were enrolled into the doubleblind lower limb study from 52 centers in 11 countries. Of these, 84 had previously received treatment with any BoNT-A (other than abobotulinumtoxinA) in their affected lower limb and were included in these post hoc analyses. Of the ITT population, 29 patients were randomized to abobotulinumtoxinA 1000 U (safety population: N = 30), 28 patients to abobotulinumtoxinA 1500 U, and 26 patients to placebo [fig_ref] Figure 1: Patient population [/fig_ref]. Baseline demographics and disease characteristics were well matched between treatment arms ( respectively. ## Patients previously treated with abobotulinumtoxinA were not included in this analysis. ## Study doses administered Overall, the median (range) abobotulinumtoxinA dose administered in the total injection volume was 1000 (867-1000) U in the 1000 U group and 1500 (800-1500) U in the 1500 U group [fig_ref] Table 2: Treatment doses administered by muscle group [/fig_ref]. Median (range) doses administered by target muscle are detailed in [fig_ref] Table 2: Treatment doses administered by muscle group [/fig_ref]. BoNT-A = botulinum toxin A; AboBoNT-A = abobotulinumtoxinA; BMI = body mass index.*Two patients received Lantox (placebo, n = 1; aboBoNT-A 1000 U, n = 0; aboBoNT-A 1500 U, n = 1) and for 11 patients the BoNT-A type was not specified (placebo, n = 1; aboBoNT-A 1000 U, n = 7; aboBoNT-A 1500 U, n = 3). Data are presented for the intention-to-treat population and reported as mean (SD) [range] unless otherwise stated. AboBoNT-A, abobotulinumtoxinA; n, number of patients who received an injection in the associated muscle group; N/A, not applicable. Data are presented for the safety population. The study protocol required that the total injection volume (7.5 mL) was divided between the soleus muscle (exactly 2.5 mL); the medial and/or lateral gastrocnemius muscle (exactly 1.5 mL); and at least one other lower limb muscle selected by the investigator (the remaining injection volume 3.5 mL split between investigator selected sites). 1), −0.9 (1.0; −3 to 0), and − 0.5 (0.7; −2 to 1) for abobotulinumtoxinA 1000 U, 1500 U, and placebo, respectively. Data at weeks 1 and 12 are shown in Supplementary Table S1 in Appendix S1. ## Change in gsc (knee extended) and soleus (knee Muscle Tone Responders (≥1 Grade Improvement in MAS Score from Baseline) for GSC (Knee Extended) and Soleus (Knee Flexed) Muscles For the GSC, a greater proportion of patients responded to treatment with abobotulinumtoxinA than placebo across time points, with a higher responder rate with 1500 U compared with 1000 U abobotulinumtoxinA at each time point [fig_ref] Figure 2: Proportion of responders [/fig_ref]. For the soleus muscle, the proportion of responders increased between weeks 1 and 4 in the abobotulinumtoxinA treatment groups, whereas responder rates in the placebo group decreased from week 1 to week 12 [fig_ref] Figure 2: Proportion of responders [/fig_ref]. For both muscle groups, the highest proportion of responders was observed at week 4 with either abobotulinumtoxinA dose. ## Gsc and soleus muscle spasticity as measured on the tardieu scale There were greater improvements in the angle of catch (X V3 ) at week 4 following abobotulinumtoxinA treatment than in the placebo arm for both the GSC and soleus muscle. The mean (SD) change was 8 (21), 6 (10), and 1 (7) for the GSC and 11 (21), 5 (9), and 0 (8) for the soleus muscle with abobotulinumtoxinA 1000 U, abobotulinumtoxinA 1500 U, and placebo, respectively. Spasticity severity (Y) decreased from baseline to week 4 for abobotulinumtoxinA 1000 U and ## Safety In total, patients previously treated with BoNT-A experienced 101 TEAEs: 36 (n = 15), 31 (n = 16), and 34 (n = 13) in the abobotulinumtoxinA 1000 U, 1500 U, and placebo groups, respectively (Supplementary [fig_ref] Table 2: Treatment doses administered by muscle group [/fig_ref] in Appendix S1). Four patients experienced a TEAE that led to study withdrawal, two in the abobotulinumtoxinA 1000 U group (pancreatic cancer, right hip pain) and one each in the 1500 U group (generalized muscle weakness, considered related to study treatment) and placebo group (loss of consciousness). The most common TEAEs for the abobotulinumtoxinA 1000 U, 1500 U, and placebo groups, respectively, were: fall (13.3%, 10.7%, and 3.8%), muscular weakness (6.7%, 14.3%, and 7.7%), and pain in an extremity (3.3%, 7.1%, and 7.7%). In total, four patients experienced four SAEs; there were two events (sinus tachycardia and pancreatic carcinoma) in the abobotulinumtoxinA 1000 U arm, one event (muscular weakness, considered to be related to study treatment) in the abobotulinumtoxinA 1500 U arm, and one event (loss of consciousness) in patients randomized to placebo. # Discussion These post hoc analyses demonstrated that a single cycle of abobotulinumtoxinA administered at either 1000 U or 1500 U reduced muscle tone and improved spasticity grade and angle in adults with spastic hemiparesis of the lower limb that had been treated previously with other BoNT-A products. Our findings support the primary results for the overall study population and confirm that abobotulinumtoxinA is an efficacious treatment option for adults with LLS. The improvements in GSC at week 4 we report here in previously treated patients are comparable with those reported for the overall trial population at each abobotulinumtoxinA dose (least squares mean [95% confidence interval]: −0.6 [−0.8, −0.5], −0.8 [−0.9, −0.7], and − 0.5 [−0.7, −0.4] for abobotulinumtoxinA 1000 U, 1500 U, and placebo, respectively). [bib_ref] Efficacy and safety of abobotulinumtoxinA in spastic lower limb: randomized trial and..., Gracies [/bib_ref] Similarly, improvements in the angle of catch (X V3 ) and a reduction in spasticity grade were in alignment with those reported in the overall study population (mean in the GSC and soleus, abobotulinumtoxinA doses combined: X V3 , +5 8 and + 5 , 8 respectively; Y, −0.3 [0.7] and − 0.4 [0.7], respectively). [bib_ref] Efficacy and safety of abobotulinumtoxinA in spastic lower limb: randomized trial and..., Gracies [/bib_ref] Compared with the previously treated patient subgroup, in the overall study population abobotulinumtoxinA dosing was similar across the medial GSC (122.5 U and 183.5 U in the 1000 U and 1500 U groups, respectively), the lateral GSC (95.2 U and 145.6 U in the 1000 U and 1500 U groups, respectively), and the soleus muscle (333.3 U and 495.3 U in the 1000 U and 1500 U groups, respectively), as reported in [fig_ref] Table 2: Treatment doses administered by muscle group [/fig_ref]. Our findings demonstrate that recommended doses of abobotulinumtoxinA (1000 U or 1500 U) were well tolerated in this patient population and that safety findings were similar to those experienced by the overall population, 3 with fall, muscular weakness, and pain in an extremity remaining the most common TEAEs. However, in the non-naïve population compared with the overall population, slightly higher rates of falls (13.3%, 10.7%, and 3.8% vs. 9.4%, 6.3%, and 3.3%, respectively, in the abobotulinumtoxinA 1000 U, 1500 U, and placebo groups, respectively) and muscular weakness (6.7%, 14.3%, and 7.7% vs 2.4%, 6.3% and 3.1%, in the abobotulinumtoxinA 1000 U, 1500 U and placebo groups, respectively) were observed. Overall, the incidence rate and type of adverse events observed here were also consistent with previous clinical studies of abobotulinumtoxinA 2 and other BoNT-A products. [bib_ref] Pooled analysis of the safety of Botulinum toxin type a in the..., Turkel [/bib_ref] These results suggest that prior treatment with other BoNT-A products does not affect abobotulinumtoxinA dosing when initiating treatment. A strength of these analyses was the sourcing of data from a phase 3, randomized, placebo-controlled study. However, as these analyses were conducted post hoc and were not statistically powered, a limitation is that we report only descriptive statistics here. Furthermore, some secondary end points from the double-blind lower limb study (such as physician's global assessment and walking speed tests) were not reported here, as it was previously shown that multiple treatment cycles were required to observe meaningful results. [bib_ref] Efficacy and safety of abobotulinumtoxinA in spastic lower limb: randomized trial and..., Gracies [/bib_ref] It should be noted that there was a large range among patients in their time since last injection with BoNT-A's other than abobotulinumtoxinA, as some patients were receiving BoNT-A treatment at regular intervals prior to this study, while others had not received treatment for a number of years allowing time for contracture, muscle shortening, compensatory gait strategies, and other confounding conditions to develop. # Conclusion These post hoc analyses demonstrate that a single cycle of treatment with abobotulinumtoxinA (1000 U or 1500 U) is associated with improvements in both muscle tone and spasticity parameters in adults with LLS previously treated with other BoNT-A products. The efficacy and safety profile of abobotulinumtoxinA in the previously treated patient subgroup was comparable to the overall study population at similar abobotulinumtoxinA doses. These findings suggest that abobotulinumtoxinA has a good efficacy and safety profile in adults with spastic hemiparesis in the lower limb who have received previous treatment with other BoNT-A products without the requirement for adjustment to recommended initial dosing. [fig] Figure 1: Patient population. AboBoNT-A, abobotulinumtoxinA; BoNT-A, botulinum toxin A; max, maximum. *Some patients had previously been treated with more than one other BoNT-A product. Patient flow is presented for the safety population. Previous BoNT-A treatments are presented for the intention-to-treat population. [/fig] [fig] Figure 2: Proportion of responders (≥1 grade improvement) to aboBoNT-A based on MAS score for (A) GSC muscle tone and (B) soleus muscle tone. AboBoNT-A, abobotulinumtoxinA; GSC, gastrocnemius-soleus complex; MAS, Modified Ashworth Scale. *At week 12, n = 1 missing in placebo and aboBoNT-A 1000 U groups, n = 2 missing in aboBoNT-A 1500 U group. Data are presented for the intention-to-treat population. Responders were defined as patients with at least a one grade improvement in muscle tone (score on the MAS) in comparison to baseline. [/fig] [table] Table 1: Mean (SD) age was 52.0 (12.8) years, and the majority (69.9%) of patients were male. Mean (SD; range) time since stroke was 5.7 (5.2; 0.9 to 27.3) years and time since traumatic brain injury was 8.1 (6.1; 1.4 to 21.0) years. The BoNT-A treatments that patients had received previously n = 13; 15.7%), and other (n = 13; 15.7%). Patients could have received more than one BoNT-A type. Doses of previous BoNT-A injections are shown inTable 1. Time from last BoNT-A injection, other than abobotulinumtoxinA, for treatment of lower limb spasticity to initiating study treatment ranged from 113 to 1769 days, 123 to 2953 days, and 127 to 2359 days, for abobotulinumtoxinA 1000 U, 1500 U, and placebo, Overall study population (double-blind phase) N = 388Patient previously treated with BoNT-A (other than aboBoNT-A) in the affected lower limb N = 84 [/table] [table] Table 2: Treatment doses administered by muscle group [/table]

The Effectiveness of Planning Interventions for Improving Physical Activity in the General Population: A Systematic Review and Meta-Analysis of Randomized Controlled Trials Planning interventions such as action planning (AP) and coping planning (CP) have been recognized as influential strategies in promoting physical activity (PA), but mixed results of existing evidence have been observed. This study aims to perform a systematical meta-analysis to evaluate the efficacy of planning interventions for improving PA in the general population. Eight databases, including Medline, Embase, PsycINFO, Cochrane Library, Web of Science, ProQuest, CNKI, and Wanfang Data, were searched to locate relevant randomized controlled trials (RCTs) from their inception to 31 December 2021. In total, 41 trials with 5439 samples were included in this systematic review, and 35 trials were used in our meta-analysis. The results showed that PA was better promoted in the planned intervention group compared to the control group (SMD = 0.35, 95% CI = 0.25-0.44, I 2 = 61.4%). Based on the subgroup analyses, we found that planning strategies were more effective among patients, males, when adopting AP intervention, when using the face-toface sessions delivery mode, and when reinforcements were conducted during the follow-up. The findings of this study indicate that planning interventions significantly improved PA behavior, and, in some contexts, the effects performed better. Future research needs to be conducted to explore the underlying mechanisms of planning interventions and validate their effects more extensively. effectiveness of PA behavior change is d = 0.27 (95% CI = 0.17 to 0.37)[11], indicating that the effect size is small but significant[12]. More recently, Whatnall et al.[13] conducted a systematic review of 66 RCT studies that examined the effects of behavioral change interventions on step-, moderate-, vigorous-intensity PA and total PA, which identified between-group differences in only 52% of the studies. Additionally, there is currently no consensus regarding which BCTs, including web-based and mobile interventions, are more effective for promoting PA[14][15][16]. To promote PA effectively, theory-based interventions that address behavioral determinants are required.Intention, an antecedent variable of behavior in the theory of planned behavior (TPB)[17], which has been taken as a crucial factor, plays a positive role in the domain of PA promotion. However, empirical studies have confirmed that there is still a gap between the formation of goal intention and PA behavior initiation[18]. Based on existed theories[19,20], Gollwitzer identified that implementation intentions, which are self-regulatory strategies, can help bridge the intention-behavior gap[21]. Health practitioners typically prefer the operational approaches of action planning (AP) and coping planning (CP) when applying implementation intention interventions, that is, to specify when, where, and how to perform behaviors and determine corresponding responses to obstacles[22].Up to now, three systematic reviews have analyzed the impact of AP and CP (or implementation intention) on the initiation and maintenance of physical activity behavior [23-25], but these studies had some limitations regarding the inclusion of high-quality literature and outcome analyses. Bélanger-Gravel et al.[23] observed a small-to-moderate overall effect size of implementation intention in several conditions through subgroup analyses. Carraro and Gaudreau [24] conducted a meta-analysis of 23 correlational and 21 experimental studies for both spontaneous and experimentally induced interventions in the form of AP and CP. This review also confirmed small-to-moderate effect sizes for planning interventions. Neither of these reviews used data entirely from RCTs as the gold standard, nor did they probe deeply into the sources of heterogeneity among studies. A recent meta-analysis [25] found significant effectiveness only in the reinforcement condition by analyzing 13 RCTs; however, its findings were limited by the number and quality of the included studies.Therefore, the main purpose of this study is to collect relevant, high-quality evidence to investigate the effectiveness of planning interventions in promoting physical activity using the method of systematic review and meta-analysis. The second purpose is to explore the sources of heterogeneity across studies through subgroup analyses and to analyze the differences in effect sizes of planning interventions across different interest variables. # Introduction Insufficient physical activity has become a public health issue globally [bib_ref] Lack of physical activity is a global problem, Haseler [/bib_ref]. Regular physical activity (PA) reduces the risk of noncommunicable diseases and all-cause mortality and delivers important prevention and treatment benefits for many different physical and psychological conditions. Nevertheless, according to a global survey of 1.9 million individuals in 168 countries, over one-quarter of people engage in minimum or no PA (150 min of moderate exercise per week or comparable) [bib_ref] Worldwide trends in insufficient physical activity from 2001 to 2016: A pooled..., Guthold [/bib_ref]. Globally, 81% of teenagers aged 11-17 years are insufficiently physically active, and older adults engage in the least amount of physical activity of all age groups [bib_ref] Psycho-social correlates of leisure-time physical activity (LTPA) among older adults: A multivariate..., Lee [/bib_ref] [bib_ref] Physical activity guidelines for older adults, Elsawy [/bib_ref]. In Canada, only 9% of children meet PA recommendations [bib_ref] Nature Elements and Fundamental Motor Skill Development Opportunities at Five Elementary School..., Lim [/bib_ref]. Thus, it is critical for public health practitioners to encourage regular PA by developing more effective interventions. Despite persistent efforts to enhance physical activity (PA) through public health policies and behavior change techniques (BCTs) [bib_ref] Effectiveness of approaches to increase physical activity behavior to prevent chronic disease..., Schwartz [/bib_ref] , interventions aimed at increasing public involvement in and adherence to PA have generated limited outcomes [bib_ref] Updating goal-setting theory in physical activity promotion: A critical conceptual review, Swann [/bib_ref] [bib_ref] Effectiveness of acceptance and commitment therapy (ACT) interventions for promoting physical activity:..., Pears [/bib_ref]. For instance, a comprehensive meta-analysis of 27 PA intervention studies found that the overall # Methods This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement [bib_ref] The PRISMA 2020 statement: An updated guideline for reporting systematic reviews, Page [/bib_ref] and Cochrane Collaboration Handbook recommendations. ## Search strategy A comprehensive literature search that has no language and publication time constraints was conducted to identify the relevant randomized controlled trials (RCTs) that probe the effectiveness of planning or implementation intention interventions on PA in the following academic databases: Medline (via PubMed), Embase, PsycINFO, Cochrane Library, Web of Science, ProQuest, Chinese National Knowledge Infrastructure (CNKI), Wanfang Data, from their inception to 31 December 2021. An exhaustive search was performed utilizing the medical subject headings (MeSH) combined with free text terms by employing Boolean logical operators, with the terms "Physical Activity", "Exercise", "Planning", "Implementation Intentions", "Action Planning", "Coping Planning", and "Randomized Controlled Trial" taken into consideration. Furthermore, as a supplementary search, we conducted a series of recursive screenings of top journals (top international journals: Health Psychology Review, Annals of Behavioral Medicine; top China journals: Sport Science, Advances in Psychological Science), grey literature, well-known publishers, and significant international academic proceedings to reduce the damage caused by the exclusion of suitable items that match our inclusion criterion. After a manual screening of the selected articles, supplementary searches were performed for other articles by important authors. In the supplement search techniques, details from all databases of search methods were displayed. All records originally retrieved were imported into EndNote 20 (Thomson ISI Research Soft, Philadelphia, PA, USA), confirmed, and managed by two authors (S.Y.P. and F.Y.) independently and concurrently. Disagreements in this process were resolved through discussion in a meeting with the other authors (A.T.O. and J.H.L.). These steps ensure the completeness and accuracy of this study. ## Eligibility criteria The following PICOS criteria were used to include relevant studies. ## Population The participants included in this study were not restricted by age, gender, health status, region, or nationality. Given that they have freedom of movement and can participate in the physical activity promotion program set up by the researchers, they are all included in the scope of this study. ## Interventions Acceptable treatments included planning interventions (AP or the combination of AP and CP), i.e., the core components of planning strategies, such as when, where, and how a goal action is to be performed or added and how to deal with the barriers to goal pursuing. Prerequisites such as an introduction to recommended amounts of PA or education on the benefits of PA were permitted in the intervention. Interventions that included other psychological treatments were excluded. ## Comparators Studies were included if the control group was set up as a waiting group with no treatments or if the control group only received some PA recommendations or education about the benefits of PA. Studies with control groups other than those were excluded. ## Outcomes Interventions that reported PA measures (e.g., number of steps, frequency of PA participation, amount of light, moderate-or high-intensity PA in time units) were included. Outcomes can be measured by any measurement instrument, including subjective selfreport questionnaires, self-administered items, and objective instruments. Outcomes must include baseline and post-test measurement data. ## Study design Only published paralleled-group RCTs were included, including pilot RCTs, with no language limitations. Duplications were deleted first, as indicated in our included and excluded criteria. The studies were independently chosen by two authors by examining their titles and abstracts. Following that, full-text reviewing was carried out to locate possibly suitable studies. Inconsistencies that arose in this section were avoided. ## Data abstraction and risk of bias assessment According to the Cochrane Consumers and Communication Review Group's data extraction template, two authors performed an independent double-blind method to investigate and extract the key data of the included studies, as follows: the first author, publication year, participants, sample sizes of different groups and total of all groups, gender, age, intervention details (strategies, duration, delivery mode, and reinforcement or not), instrument and various outcomes, etc. Missing data were obtained by emailing the corresponding author of the related study or retrieving other systematic reviews that included the studies. Based on the Cochrane Risk of Bias tool, seven items were structured to assess the risk of bias for the included studies: (1) random sequence generation: whether the random sequence method and process are described in detail; (2) allocation concealment: whether to allocate participants strictly according to the results of random numbers; (3) blinding of participants: whether subjects and investigators were blinded; (4) blinding of outcome assessment: whether the evaluators were blinded; (5) incomplete outcome data: whether the drop-out rate was controlled within 10%; (6) selective data reporting: whether only favorable results were selectively reported; (7) other undefined biases: bias such as conflict of interest, small sample size, and baseline imbalance. Each of the items was graded as high risk of bias, low risk of bias, and unknown risk of bias, respectively. The study was assessed as low risk if more than four items were low risk. If most of the study was unknown, the study was assessed as unknown. When more than one item was high risk and less than four other items were rated as low risk, the study was considered high risk. Two reviewers independently assessed the bias risk of the included literature, and disagreements were judged by a third reviewer. ## Statistical analyses According to Cochrane Collaboration Handbook recommendations, the present meta-analysis was conducted by a conventional pair of crossed trials for each comparison at post-intervention. To begin, the omnibus homogeneity test (Q) and I 2 statistics were used to assess study heterogeneity, and p < 0.1 for Q was regarded as statistically significant, while I 2 statistics had values of 25%, 50%, and 75%, indicating mild, moderate, and high heterogeneity [bib_ref] Quantifying heterogeneity in a meta-analysis, Higgins [/bib_ref]. Second, means and standard deviations of outcomes reported postintervention were uniformly extracted as numerical variables for meta-analysis to ensure more accurate analysis outcomes. Moreover, to evaluate the primary effect of planning interventions, the effect sizes were pooled using the inverse variance statistical approach with random effect models. The pooled effect sizes were provided as standardized mean differences (SMDs) with respective 95% confidence intervals (CIs). SMDs were calculated by the mean and standard deviation of each comparison group. When standard errors and confidence intervals were reported instead of standard deviations, they were transformed according to statistical methods. Third, comparison-adjusted funnel plots were drawn to visually detect various forms of potential publication bias. Egger's test was used as a supplement to the quantitative evaluation of the funnel plot to test the significance level [bib_ref] How important are comprehensive literature searches and the assessment of trial quality..., Egger [/bib_ref]. Then, a sensitivity analysis was performed, removing the studies deemed to be at high risk of bias. Lastly, given that the role of moderator variables may be the source of heterogeneity between studies, subgroup analyses were performed to ensure the stability of the overall effect sizes. The subgroup analysis performed in this study is shown as follows: Intervention Strategy (AP vs. AP + CP); Duration (≥5 weeks vs. <5 weeks); Publication Year (year ≥ 2012 vs. year < 2012). Delivery Mode (Sessions vs. Online vs. Sessions and Online); Reinforcement or not (Yes vs. No); Participants (Patients vs. Healthy Populations); Students or not (Yes or No); Female-to-Male Ratio (≥1 vs. <1); Sample Size (≥100 vs. <100); Instrument (Objective vs. Self-report vs. Items). The above analyses were carried out in STATA 14.1 (StataCorp, College Station, TX, USA). # Results ## Literature selection The initial database search yielded a total of 1790 records. Before screening, 374 duplicate records and 794 records with only simple protocols were removed. After the first round of careful screening of titles and abstracts, 254 search records entered the next step for re-examination. After 131 records were excluded, 123 studies were left for full-text review. As a result, 41 articles were included in this systematic review, and only six articles [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] [bib_ref] Effect of implementation intention on walking in people with diabetes: An experimental..., Vieira Da Silva [/bib_ref] [bib_ref] The Effect of Integrated Intervention Based on Protection Motivation Theory and Implementation..., Morowatisharifabad [/bib_ref] were not suitable for quantitative meta-analysis. The selection process is shown in [fig_ref] Figure 1: Flow chart of study design by PRISMA 2020 [/fig_ref]. ## Literature selection The initial database search yielded a total of 1790 records. Before screening, 37 plicate records and 794 records with only simple protocols were removed. After the round of careful screening of titles and abstracts, 254 search records entered the nex for re-examination. After 131 records were excluded, 123 studies were left for ful review. As a result, 41 articles were included in this systematic review, and six articles [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] [bib_ref] Effect of implementation intention on walking in people with diabetes: An experimental..., Vieira Da Silva [/bib_ref] [bib_ref] The Effect of Integrated Intervention Based on Protection Motivation Theory and Implementation..., Morowatisharifabad [/bib_ref] were not suitable for quantitative meta-analysis. The s tion process is shown in [fig_ref] Figure 1: Flow chart of study design by PRISMA 2020 [/fig_ref]. [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] [bib_ref] Mental contrasting and implementation intentions to increase physical activity in sedentary, disadvantaged..., Abbott [/bib_ref] , and 3 studies that did not report this cond [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] [bib_ref] Mental contrasting and implementation intentions to increase physical activity in sedentary, disadvantaged..., Abbott [/bib_ref] , and 3 studies that did not report this condition [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] , with publication years ranging from 2002 to 2021. All studies were published in English except for one study published in Chinese [bib_ref] The Promotion Strategies of Pupils' Exercise Intention and Behavior:the Action Planning Intervention..., Xiang [/bib_ref] and another in Spanish [bib_ref] Impact of the activation of intention to perform physical activity in type..., Silva [/bib_ref]. In total, 2936 participants were randomized to the planning interventions group, while 2948 participants were assigned to the controlled group, with the mean age ranging between 8.06 and 73.30 years old. There were 5 studies targeting women [bib_ref] Helping middle-aged women translate physical activity intentions into action: Combining the theory..., Arbour [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] Examining implementation intentions in an exercise intervention: The effects on adherence and..., Murray [/bib_ref] [bib_ref] Physical activity in women: Effects of a self-regulation intervention, Stadler [/bib_ref] [bib_ref] Implementation intentions for physical activity behavior in older adult women: An examination..., Hall [/bib_ref] and 28 studies with more than 50% female participants [bib_ref] Combining motivational and volitional interventions to promote exercise participation: Protection motivation theory..., Milne [/bib_ref] [bib_ref] The use of implementation intentions and the decision balance sheet in promoting..., Prestwich [/bib_ref] [bib_ref] Helping middle-aged women translate physical activity intentions into action: Combining the theory..., Arbour [/bib_ref] [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] The Effects of Self-Discordance, Self-Concordance, and Implementation Intentions on Health Behavior, Chatzisarantis [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] Should implementation intentions interventions be implemented in obesity prevention: The impact of..., Vet [/bib_ref] [bib_ref] Changing nutrition, physical activity and body weight among student nurses and midwives:..., Luszczynska [/bib_ref] [bib_ref] Examining implementation intentions in an exercise intervention: The effects on adherence and..., Murray [/bib_ref] [bib_ref] Can the effects of implementation intentions on exercise be enhanced using text..., Prestwich [/bib_ref] [bib_ref] Physical activity in women: Effects of a self-regulation intervention, Stadler [/bib_ref] [bib_ref] Effect of Implementation Intentions to Change Behaviour: Moderation by Intention Stability, Godin [/bib_ref] [bib_ref] Can implementation intentions and text messages promote brisk walking? A randomized trial, Prestwich [/bib_ref] [bib_ref] Imagery and implementation intention: A randomised controlled trial of interventions to increase..., Andersson [/bib_ref] [bib_ref] Self-efficacy as a moderator of the planning-behaviour relationship in interventions designed to..., Luszczynska [/bib_ref] [bib_ref] Araújo-Soares, V. Do brief online planning interventions increase physical activity amongst university..., Skår [/bib_ref] [bib_ref] A combined planning and self-efficacy intervention to promote physical activity: A multiple..., Koring [/bib_ref] [bib_ref] Randomized Controlled Trial of Collaborative Implementation Intentions Targeting Working Adults' Physical Activity, Prestwich [/bib_ref] [bib_ref] Implementation intentions for physical activity behavior in older adult women: An examination..., Hall [/bib_ref] [bib_ref] Impact of the activation of intention to perform physical activity in type..., Silva [/bib_ref] [bib_ref] An intervention crossing two types of action planning with mental simulation for..., Koka [/bib_ref] [bib_ref] Revisiting self-regulatory techniques to promote physical activity in older adults: Null-findings from..., Warner [/bib_ref] [bib_ref] Mental contrasting and implementation intentions to increase physical activity in sedentary, disadvantaged..., Abbott [/bib_ref] [bib_ref] Which behaviour change techniques are effective to promote physical activity and reduce..., Schroé [/bib_ref] [bib_ref] Effect of implementation intention on walking in people with diabetes: An experimental..., Vieira Da Silva [/bib_ref] [bib_ref] Collaborative, dyadic, and individual planning and physical activity: A dyadic randomized controlled..., Kulis [/bib_ref] [bib_ref] The Effect of Integrated Intervention Based on Protection Motivation Theory and Implementation..., Morowatisharifabad [/bib_ref]. [fig_ref] Table 1: Characteristics of studies [/fig_ref] provides a detailed introduction to these demographic characteristics. As regards the type of planning strategies chosen by the PA promotion intervention, the number of studies that chose AP (n = 21) as an intervention strategy and the number of studies that chose AP combined with CP (n = 20) as an intervention strategy were very similar. ## Characteristics of studies ## Characteristics of studies The delivery of planning interventions was characterized by three typical modes. The first mode (n = 24) is to implement the intervention through face-to-face individual or group sessions; the second (n = 5) is to conduct online delivery modes, such as phone calls, emails, postal mail, pedometers, phone text messages, APP tracking, and website feedback; and the third mode (n = 12) is to combine face-to-face sessions with online delivery. The duration of the intervention (from baseline to the last endpoint) of the included studies ranged from 1 week to 14 months. After the baseline interventions were applied, some studies reinforced the effects of the baseline interventions through telephone surveys, text message reminders, diary records, and face-to-face sessions. Across all studies, 20 studies used standardized self-report questionnaires tested for reliability and the validity of previous studies to assess physical activity, 15 studies employed measurement items (three of which were validated), 5 studies used objective instruments (two accelerometers and two pedometers), 1 study used diaries, and 1 study used checklists. In most of the studies, the control groups received some motivational education, including the benefits of physical activity and WHO physical activity recommendations through face-to-face sessions, leaflets, emails, or text messages. This motivational education was also implemented simultaneously in the intervention group. Moreover, intervention completers were considered for statistical analysis in most studies and only eleven studies employed an intention-to-treat approach [bib_ref] Physical activity and depressive symptoms in cardiac rehabilitation: Long-term effects of a..., Scholz [/bib_ref] [bib_ref] Changing nutrition, physical activity and body weight among student nurses and midwives:..., Luszczynska [/bib_ref] [bib_ref] Physical activity in women: Effects of a self-regulation intervention, Stadler [/bib_ref] [bib_ref] Beyond good intentions: The role of proactive coping in achieving sustained behavioural..., Thoolen [/bib_ref] [bib_ref] The roles of behavioral and implementation intentions in changing physical activity in..., Armitage [/bib_ref] [bib_ref] Self-efficacy as a moderator of the planning-behaviour relationship in interventions designed to..., Luszczynska [/bib_ref] [bib_ref] Araújo-Soares, V. Do brief online planning interventions increase physical activity amongst university..., Skår [/bib_ref] [bib_ref] Implementation intentions for physical activity behavior in older adult women: An examination..., Hall [/bib_ref] [bib_ref] An intervention crossing two types of action planning with mental simulation for..., Koka [/bib_ref] [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] [bib_ref] Collaborative, dyadic, and individual planning and physical activity: A dyadic randomized controlled..., Kulis [/bib_ref]. ## Quality of included studies In 34 of the included trials [bib_ref] The use of implementation intentions and the decision balance sheet in promoting..., Prestwich [/bib_ref] [bib_ref] Helping middle-aged women translate physical activity intentions into action: Combining the theory..., Arbour [/bib_ref] [bib_ref] Long-term effects of two psychological interventions on physical exercise and self-regulation following..., Sniehotta [/bib_ref] [bib_ref] The efficacy of an implementation intention intervention for promoting physical activity among..., Latimer [/bib_ref] [bib_ref] An implementation intentions intervention, the use of a planning strategy, and physical..., Luszczynska [/bib_ref] [bib_ref] Physical activity and depressive symptoms in cardiac rehabilitation: Long-term effects of a..., Scholz [/bib_ref] [bib_ref] Action plans and coping plans for physical exercise: A longitudinal intervention study..., Sniehotta [/bib_ref] [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] The Effects of Self-Discordance, Self-Concordance, and Implementation Intentions on Health Behavior, Chatzisarantis [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] Should implementation intentions interventions be implemented in obesity prevention: The impact of..., Vet [/bib_ref] [bib_ref] Changing nutrition, physical activity and body weight among student nurses and midwives:..., Luszczynska [/bib_ref] [bib_ref] Examining implementation intentions in an exercise intervention: The effects on adherence and..., Murray [/bib_ref] [bib_ref] Can the effects of implementation intentions on exercise be enhanced using text..., Prestwich [/bib_ref] [bib_ref] Physical activity in women: Effects of a self-regulation intervention, Stadler [/bib_ref] [bib_ref] The roles of behavioral and implementation intentions in changing physical activity in..., Armitage [/bib_ref] [bib_ref] Can implementation intentions and text messages promote brisk walking? A randomized trial, Prestwich [/bib_ref] [bib_ref] Imagery and implementation intention: A randomised controlled trial of interventions to increase..., Andersson [/bib_ref] [bib_ref] Self-efficacy as a moderator of the planning-behaviour relationship in interventions designed to..., Luszczynska [/bib_ref] [bib_ref] Araújo-Soares, V. Do brief online planning interventions increase physical activity amongst university..., Skår [/bib_ref] [bib_ref] Randomized Controlled Trial of Collaborative Implementation Intentions Targeting Working Adults' Physical Activity, Prestwich [/bib_ref] [bib_ref] Using a combined motivational and volitional intervention to promote exercise and healthy..., Zhang [/bib_ref] [bib_ref] Randomized controlled trial of a behavior change intervention to increase physical activity..., Mcgowan [/bib_ref] [bib_ref] Moving Heart Program": An intervention to improve physical activity among patients with..., Rodrigues [/bib_ref] [bib_ref] Implementation intentions for physical activity behavior in older adult women: An examination..., Hall [/bib_ref] [bib_ref] Impact of the activation of intention to perform physical activity in type..., Silva [/bib_ref] [bib_ref] Revisiting self-regulatory techniques to promote physical activity in older adults: Null-findings from..., Warner [/bib_ref] [bib_ref] The Promotion Strategies of Pupils' Exercise Intention and Behavior:the Action Planning Intervention..., Xiang [/bib_ref] [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] [bib_ref] Which behaviour change techniques are effective to promote physical activity and reduce..., Schroé [/bib_ref] [bib_ref] Effect of implementation intention on walking in people with diabetes: An experimental..., Vieira Da Silva [/bib_ref] [bib_ref] Collaborative, dyadic, and individual planning and physical activity: A dyadic randomized controlled..., Kulis [/bib_ref] [bib_ref] The Effect of Integrated Intervention Based on Protection Motivation Theory and Implementation..., Morowatisharifabad [/bib_ref] , the risk of bias was classified as low or uncertain, whereas 7 studies [bib_ref] Combining motivational and volitional interventions to promote exercise participation: Protection motivation theory..., Milne [/bib_ref] [bib_ref] Beyond good intentions: The role of proactive coping in achieving sustained behavioural..., Thoolen [/bib_ref] [bib_ref] Effect of Implementation Intentions to Change Behaviour: Moderation by Intention Stability, Godin [/bib_ref] [bib_ref] A combined planning and self-efficacy intervention to promote physical activity: A multiple..., Koring [/bib_ref] [bib_ref] An intervention crossing two types of action planning with mental simulation for..., Koka [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] [bib_ref] Mental contrasting and implementation intentions to increase physical activity in sedentary, disadvantaged..., Abbott [/bib_ref] were classified with high-risk bias. In all 41 studies, sufficient random sequence generation was observed, whereas few of them had conducted their allocation concealment. Three studies [bib_ref] Can implementation intentions and text messages promote brisk walking? A randomized trial, Prestwich [/bib_ref] [bib_ref] Using a combined motivational and volitional intervention to promote exercise and healthy..., Zhang [/bib_ref] [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] explicitly mentioned a sufficient blinding process of participants and researchers, whereas others were unclear. Relatively complete outcome analyses and reports were shown in most of the studies, except in nine studies [bib_ref] Combining motivational and volitional interventions to promote exercise participation: Protection motivation theory..., Milne [/bib_ref] [bib_ref] Beyond good intentions: The role of proactive coping in achieving sustained behavioural..., Thoolen [/bib_ref] [bib_ref] Effect of Implementation Intentions to Change Behaviour: Moderation by Intention Stability, Godin [/bib_ref] [bib_ref] Araújo-Soares, V. Do brief online planning interventions increase physical activity amongst university..., Skår [/bib_ref] [bib_ref] A combined planning and self-efficacy intervention to promote physical activity: A multiple..., Koring [/bib_ref] [bib_ref] Randomized Controlled Trial of Collaborative Implementation Intentions Targeting Working Adults' Physical Activity, Prestwich [/bib_ref] [bib_ref] Using a combined motivational and volitional intervention to promote exercise and healthy..., Zhang [/bib_ref] [bib_ref] An intervention crossing two types of action planning with mental simulation for..., Koka [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] with relatively high drop-out rates. Regarding other bias factors, three studies [bib_ref] Imagery and implementation intention: A randomised controlled trial of interventions to increase..., Andersson [/bib_ref] [bib_ref] Impact of the activation of intention to perform physical activity in type..., Silva [/bib_ref] [bib_ref] Mental contrasting and implementation intentions to increase physical activity in sedentary, disadvantaged..., Abbott [/bib_ref] were deemed to have a high-risk bias. [fig_ref] Figure 2: Risk of bias graph. [/fig_ref] show details on overall and individual quality. The delivery of planning interventions was characterized by three typical modes. The first mode (n = 24) is to implement the intervention through face-to-face individual or group sessions; the second (n = 5) is to conduct online delivery modes, such as phone calls, emails, postal mail, pedometers, phone text messages, APP tracking, and website feedback; and the third mode (n = 12) is to combine face-to-face sessions with online delivery. The duration of the intervention (from baseline to the last endpoint) of the included studies ranged from 1 week to 14 months. After the baseline interventions were applied, some studies reinforced the effects of the baseline interventions through telephone surveys, text message reminders, diary records, and face-to-face sessions. Across all studies, 20 studies used standardized self-report questionnaires tested for reliability and the validity of previous studies to assess physical activity, 15 studies employed measurement items (three of which were validated), 5 studies used objective instruments (two accelerometers and two pedometers), 1 study used diaries, and 1 study used checklists. In most of the studies, the control groups received some motivational education, including the benefits of physical activity and WHO physical activity recommendations through face-to-face sessions, leaflets, emails, or text messages. This motivational education was also implemented simultaneously in the intervention group. Moreover, intervention completers were considered for statistical analysis in most studies and only eleven studies employed an intention-to-treat approach [bib_ref] Physical activity and depressive symptoms in cardiac rehabilitation: Long-term effects of a..., Scholz [/bib_ref] [bib_ref] Changing nutrition, physical activity and body weight among student nurses and midwives:..., Luszczynska [/bib_ref] [bib_ref] Physical activity in women: Effects of a self-regulation intervention, Stadler [/bib_ref] [bib_ref] Beyond good intentions: The role of proactive coping in achieving sustained behavioural..., Thoolen [/bib_ref] [bib_ref] The roles of behavioral and implementation intentions in changing physical activity in..., Armitage [/bib_ref] [bib_ref] Self-efficacy as a moderator of the planning-behaviour relationship in interventions designed to..., Luszczynska [/bib_ref] [bib_ref] Araújo-Soares, V. Do brief online planning interventions increase physical activity amongst university..., Skår [/bib_ref] [bib_ref] Implementation intentions for physical activity behavior in older adult women: An examination..., Hall [/bib_ref] [bib_ref] An intervention crossing two types of action planning with mental simulation for..., Koka [/bib_ref] [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] [bib_ref] Collaborative, dyadic, and individual planning and physical activity: A dyadic randomized controlled..., Kulis [/bib_ref]. ## Quality of included studies In 34 of the included trials [bib_ref] The use of implementation intentions and the decision balance sheet in promoting..., Prestwich [/bib_ref] [bib_ref] Helping middle-aged women translate physical activity intentions into action: Combining the theory..., Arbour [/bib_ref] [bib_ref] Long-term effects of two psychological interventions on physical exercise and self-regulation following..., Sniehotta [/bib_ref] [bib_ref] The efficacy of an implementation intention intervention for promoting physical activity among..., Latimer [/bib_ref] [bib_ref] An implementation intentions intervention, the use of a planning strategy, and physical..., Luszczynska [/bib_ref] [bib_ref] Physical activity and depressive symptoms in cardiac rehabilitation: Long-term effects of a..., Scholz [/bib_ref] [bib_ref] Action plans and coping plans for physical exercise: A longitudinal intervention study..., Sniehotta [/bib_ref] [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] The Effects of Self-Discordance, Self-Concordance, and Implementation Intentions on Health Behavior, Chatzisarantis [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] Should implementation intentions interventions be implemented in obesity prevention: The impact of..., Vet [/bib_ref] [bib_ref] Changing nutrition, physical activity and body weight among student nurses and midwives:..., Luszczynska [/bib_ref] [bib_ref] Examining implementation intentions in an exercise intervention: The effects on adherence and..., Murray [/bib_ref] [bib_ref] Can the effects of implementation intentions on exercise be enhanced using text..., Prestwich [/bib_ref] [bib_ref] Physical activity in women: Effects of a self-regulation intervention, Stadler [/bib_ref] [bib_ref] The roles of behavioral and implementation intentions in changing physical activity in..., Armitage [/bib_ref] [bib_ref] Can implementation intentions and text messages promote brisk walking? A randomized trial, Prestwich [/bib_ref] [bib_ref] Imagery and implementation intention: A randomised controlled trial of interventions to increase..., Andersson [/bib_ref] [bib_ref] Self-efficacy as a moderator of the planning-behaviour relationship in interventions designed to..., Luszczynska [/bib_ref] [bib_ref] Araújo-Soares, V. Do brief online planning interventions increase physical activity amongst university..., Skår [/bib_ref] [bib_ref] Randomized Controlled Trial of Collaborative Implementation Intentions Targeting Working Adults' Physical Activity, Prestwich [/bib_ref] [bib_ref] Using a combined motivational and volitional intervention to promote exercise and healthy..., Zhang [/bib_ref] [bib_ref] Randomized controlled trial of a behavior change intervention to increase physical activity..., Mcgowan [/bib_ref] [bib_ref] Moving Heart Program": An intervention to improve physical activity among patients with..., Rodrigues [/bib_ref] [bib_ref] Implementation intentions for physical activity behavior in older adult women: An examination..., Hall [/bib_ref] [bib_ref] Impact of the activation of intention to perform physical activity in type..., Silva [/bib_ref] [bib_ref] Revisiting self-regulatory techniques to promote physical activity in older adults: Null-findings from..., Warner [/bib_ref] [bib_ref] The Promotion Strategies of Pupils' Exercise Intention and Behavior:the Action Planning Intervention..., Xiang [/bib_ref] [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] [bib_ref] Which behaviour change techniques are effective to promote physical activity and reduce..., Schroé [/bib_ref] [bib_ref] Effect of implementation intention on walking in people with diabetes: An experimental..., Vieira Da Silva [/bib_ref] [bib_ref] Collaborative, dyadic, and individual planning and physical activity: A dyadic randomized controlled..., Kulis [/bib_ref] [bib_ref] The Effect of Integrated Intervention Based on Protection Motivation Theory and Implementation..., Morowatisharifabad [/bib_ref] , the risk of bias was classified as low or uncertain, whereas 7 studies [bib_ref] Combining motivational and volitional interventions to promote exercise participation: Protection motivation theory..., Milne [/bib_ref] [bib_ref] Beyond good intentions: The role of proactive coping in achieving sustained behavioural..., Thoolen [/bib_ref] [bib_ref] Effect of Implementation Intentions to Change Behaviour: Moderation by Intention Stability, Godin [/bib_ref] [bib_ref] A combined planning and self-efficacy intervention to promote physical activity: A multiple..., Koring [/bib_ref] [bib_ref] An intervention crossing two types of action planning with mental simulation for..., Koka [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] [bib_ref] Mental contrasting and implementation intentions to increase physical activity in sedentary, disadvantaged..., Abbott [/bib_ref] were classified with high-risk bias. In all 41 studies, sufficient random sequence generation was observed, whereas few of them had conducted their allocation concealment. Three studies [bib_ref] Can implementation intentions and text messages promote brisk walking? A randomized trial, Prestwich [/bib_ref] [bib_ref] Using a combined motivational and volitional intervention to promote exercise and healthy..., Zhang [/bib_ref] [bib_ref] A randomized controlled trial of WATAAP to promote physical activity in colorectal..., Maxwell-Smith [/bib_ref] explicitly mentioned a sufficient blinding process of participants and researchers, whereas others were unclear. Relatively complete outcome analyses and reports were shown in most of the studies, except in nine studies [bib_ref] Combining motivational and volitional interventions to promote exercise participation: Protection motivation theory..., Milne [/bib_ref] [bib_ref] Beyond good intentions: The role of proactive coping in achieving sustained behavioural..., Thoolen [/bib_ref] [bib_ref] Effect of Implementation Intentions to Change Behaviour: Moderation by Intention Stability, Godin [/bib_ref] [bib_ref] Araújo-Soares, V. Do brief online planning interventions increase physical activity amongst university..., Skår [/bib_ref] [bib_ref] A combined planning and self-efficacy intervention to promote physical activity: A multiple..., Koring [/bib_ref] [bib_ref] Randomized Controlled Trial of Collaborative Implementation Intentions Targeting Working Adults' Physical Activity, Prestwich [/bib_ref] [bib_ref] Using a combined motivational and volitional intervention to promote exercise and healthy..., Zhang [/bib_ref] [bib_ref] An intervention crossing two types of action planning with mental simulation for..., Koka [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] with relatively high dropout rates. Regarding other bias factors, three studies [bib_ref] Imagery and implementation intention: A randomised controlled trial of interventions to increase..., Andersson [/bib_ref] [bib_ref] Impact of the activation of intention to perform physical activity in type..., Silva [/bib_ref] [bib_ref] Mental contrasting and implementation intentions to increase physical activity in sedentary, disadvantaged..., Abbott [/bib_ref] were deemed to have a high-risk bias. [fig_ref] Figure 2: Risk of bias graph. [/fig_ref] show details on overall and individual quality. ## Primary outcome Overall effect sizes were combined for the 35 trials included in the meta-analysis; 6 studies [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] [bib_ref] Effect of implementation intention on walking in people with diabetes: An experimental..., Vieira Da Silva [/bib_ref] [bib_ref] The Effect of Integrated Intervention Based on Protection Motivation Theory and Implementation..., Morowatisharifabad [/bib_ref] were excluded due to unavailable data. All studies used the measurement of PA as the outcome indicator. By means of the random effect model, the planning interventions group yielded a small-to-medium significant pooled effect size (SMD = 0.35, 95% CI: 0.25, 0.44, I 2 = 61.4%) compared to the controlled group (shown in [fig_ref] Table 2: Primary results and subgroup analyses [/fig_ref]. The effect sizes varied between −0.12 and 0.94 across the studies. A funnel plot indicated that there was no publication bias (shown in [fig_ref] Figure 1: Flow chart of study design by PRISMA 2020 [/fig_ref] , but the quantitative Egger test did not reveal the same result (p-value = 0.001) (shown in [fig_ref] Figure 2: Risk of bias graph. [/fig_ref]. ## Primary outcome Overall effect sizes were combined for the 35 trials included in the meta-analysis; 6 studies [bib_ref] Implementation intentions, occupational stress, and the exercise intention-behavior relationship, Budden [/bib_ref] [bib_ref] A randomised controlled trial of the effects of implementation intentions on women's..., Arbour [/bib_ref] [bib_ref] A brief intervention to increase physical activity behavior among adolescents using mental..., Koka [/bib_ref] [bib_ref] Effect of implementation intention on walking in people with diabetes: An experimental..., Vieira Da Silva [/bib_ref] [bib_ref] The Effect of Integrated Intervention Based on Protection Motivation Theory and Implementation..., Morowatisharifabad [/bib_ref] were excluded due to unavailable data. All studies used the measurement of PA as the outcome indicator. By means of the random effect model, the planning interventions group yielded a small-to-medium significant pooled effect size (SMD = 0.35, 95% CI: 0.25, 0.44, I 2 = 61.4%) compared to the controlled group (shown in [fig_ref] Table 2: Primary results and subgroup analyses [/fig_ref]. The effect sizes varied between −0.12 and 0.94 across the studies. A funnel plot indicated that there was no publication bias (shown in [fig_ref] Figure 1: Flow chart of study design by PRISMA 2020 [/fig_ref] , but the quantitative Egger test did not reveal the same result (p-value = 0.001) (shown in [fig_ref] Figure 2: Risk of bias graph. [/fig_ref]. ## Subgroup analyses The results of the predefined subgroup analyses, separated into ten subgroups, are shown in [fig_ref] Table 2: Primary results and subgroup analyses [/fig_ref] # Discussion Thirty-five high-quality RCTs were included in the present study for meta-analysis, and the results found that the planning strategies intervention significantly promoted physical activity in the general population, with the overall effect size (SMD = 0.35, 95% CI: 0.25, 0.44) being "small-to-medium" according to Cohen's classification criteria of effect size . Subgroup analyses were conducted and revealed that the planning interventions were more effective in the patient group, the group with fewer females. Moreover, the delivery mode of individual or group face-to-face sessions during the imposition of the intervention and the group that underwent post-intervention reinforcement performed better. We also found that the effects of different measurement instruments and sample sizes on the pooled effect sizes suggested that they may be sources of heterogeneity between studies. A positive and significant intervention effect, as revealed by this study, identified that planning strategies can improve PA successfully. Bélanger-Gravel et al. [bib_ref] A meta-analytic review of the effect of implementation intentions on physical activity, Belanger-Gravel [/bib_ref] conducted the first meta-analysis of an AP-induced trial, showing that the planning intervention had a significant effect on physical activity, both post-intervention and at follow-up. Almost simultaneously, Carraro and Gaudreau [bib_ref] Spontaneous and experimentally induced action planning and coping planning for physical activity:..., Carraro [/bib_ref] also conducted a meta-analysis combining data from both correlational and experimental studies; it showed that both spontaneous and experimentally induced AP and CP were successful in promoting physical activity. A recent meta-analysis of BCT interventions incorporating AP, conducted by Howlett et al. [bib_ref] Are physical activity interventions for healthy inactive adults effective in promoting behavior..., Howlett [/bib_ref] , also showed a significant small-to-moderate effect size effect of BCTs on initiating PA behaviors. This study, the largest meta-analysis of high-quality RCTs to date, further validated the significant effect of planning interventions to promote physical activity, which results from the key role that planning strategies play in behavioral change as self-regulatory strategies [bib_ref] Implementation intention and action planning interventions in health contexts: State of the..., Hagger [/bib_ref]. According to the health action process approach (HAPA), two types of planning strategies, AP and CP, play a key role in the initiation and maintenance of intended behavior [bib_ref] Modeling health behavior change: How to predict and modify the adoption and..., Schwarzer [/bib_ref]. The function of AP is to enhance awareness in the individual about potential future scenarios in which the behavior may be performed by making clear when, when, and how the individual would initiate the behavior [bib_ref] Health Action Process Approach (HAPA) as a Theoretical Framework to Understand Behavior..., Schwarzer [/bib_ref]. CP focuses on the anticipation of barriers that may interfere with a desired activity and how to choose alternative behaviors that may be implemented to overcome those barriers [bib_ref] Increasing physical exercise levels: Age-specific benefits of planning, Scholz [/bib_ref] [bib_ref] A meta-analysis of the health action process approach, Zhang [/bib_ref]. As mental simulations of a series of behavioral processes, planning strategies facilitate the successful translation of good intentions into action through the pre-construction of situations that initiate behavior and the management of possible anticipated obstacles [bib_ref] From intentions via planning and behavior to physical exercise habits, Fleig [/bib_ref] [bib_ref] Psychological mechanisms in a digital intervention to improve physical activity: A multicentre..., Schwarzer [/bib_ref]. Moreover, AP and CP have been designed in HAPA as mediating variables between intentions and behaviors, helping to bridge the gap between intention and behavior [bib_ref] Bridging the intention-behaviour gap: Planning, self-efficacy, and action control in the adoption..., Sniehotta [/bib_ref]. Some studies have empirically confirmed that AP and CP can also moderate the relationship between intention and behavior [bib_ref] The theory of planned behavior and exercise: Evidence for the mediating and..., Norman [/bib_ref] [bib_ref] Mechanisms of health behavior change in persons with chronic illness or disability:..., Schwarzer [/bib_ref] [bib_ref] How does the health action process approach (HAPA) bridge the intention-behavior gap?..., Sutton [/bib_ref]. From the above analysis, it can be identified that AP and CP are crucial psychological determinants of PA initiation, and future research should explore the deeper mechanisms of action of AP and CP in promoting PA [bib_ref] Planning and implementation intention interventions in physical activity, Moyers [/bib_ref]. The exploratory subgroup analyses conducted in this study revealed that the effects of the planning interventions differed across conditions and contexts, which contributes to a cautious interpretation of the overall effect sizes. In the intervention strategy group, the intervention effects of AP were superior to AP combined with CP, which may stem from the fact that the AP conducted in the trials was more acceptable to the participants, while the combined strategy added an extra CP to the psychological process of behavior change using an if-then format "cue" in response to behavior obstacles [bib_ref] Spontaneous and experimentally induced action planning and coping planning for physical activity:..., Carraro [/bib_ref] , which may have led to a decline of intervention effects. However, the combination of AP and CP is also a promising choice of an effective strategy for increasing PA, and its efficacy needs to be verified by more RCTs. Moreover, in terms of intervention delivery modes, face-to-face sessions were the most effective, with online sessions alone (e.g., telephone calls, emails, or visiting websites) being the least effective; post-intervention groups with reinforcement achieved better results. As self-regulatory strategies, planning strategies need to control the details of the interventions to be effective in promoting complex behaviors such as physical activity, so the interventions were more effective in the cases using the delivery mode of face-to-face sessions that were adept in focusing the participants' attention and the addition of reinforcements during the follow-up period. Subgroup analysis by publication year showed a higher effect size for studies published before 2012 than those published after 2012, indicating a decreasing trend in overall effect sizes for studies in the last decade; this trend needed to be verified by more evidence. Furthermore, in the subgroup analysis of the different samples, it was found that the planned interventions were more effective with the patients than with the healthy population, which supports the idea that planning interventions were important interventions for rehabilitating patients [bib_ref] Effectiveness of physical activity interventions delivered or prompted by health professionals in..., Kettle [/bib_ref]. In addition, the interventions were less effective in the population with a high proportion of females, which may be more related to the intention status of the study sample. The planning interventions had a better effect among those with PA intention [bib_ref] Matched or nonmatched interventions based on the transtheoretical model to promote physical..., Romain [/bib_ref] [bib_ref] Using action planning to promote exercise behavior, Conner [/bib_ref] , while most females are usually unintentional due to a lack of interest in PA. Although no visible difference was observed between the student and non-student groups, planning strategies remain promising interventions to promote students participating in PA because of their low cost and ease of implementation in campus settings. Of note, the results from the instrument group in the subgroup analyses suggest that differences between instruments may have contributed to the heterogeneity of the studies. Future studies should employ validated instruments of PA, such as objective instruments (e.g., accelerometers and pedometers) or widely recognized self-report questionnaires (e.g., LTEQ [bib_ref] A simple method to assess exercise behavior in the community, Godin [/bib_ref] and IPAQ [bib_ref] International physical activity questionnaire: 12-country reliability and validity, Craig [/bib_ref]. Given that objective instruments and self-reported questionnaires measure the different parts of PA and that such measurement outcomes are not equivalent [bib_ref] Evolution of accelerometer methods for physical activity research, Troiano [/bib_ref] , further investigation of more appropriate approaches to merging objective instruments and self-reported questionnaires would contribute to improving the validity of evidence based on PA measurements. The present study is the first meta-analysis of planning interventions for PA that uses RCTs and includes a significant amount of literature covering a wide range of populations (mean age from 8.06 to 73.30 years old). Although only 11 studies used intention-totreat analysis as a method of calculating outcome indicators, the inclusion of far more than 15 trials gives credibility to the outcomes [bib_ref] Effect sizes for continuous data, Borenstein [/bib_ref]. Based on the high-quality literature included and the rigorous research procedure, the findings of this study elucidate the broad effectiveness of planning interventions. As low-cost interventions that can be delivered in a variety of ways, planning interventions can be easily disseminated and promoted to a wide range of populations, providing them with promising strategies used in the public health domain to increase physical activity and prevent noncommunicable diseases caused by sedentary and physical inactivity. Several uncontrollable limitations also affected the results of this study. Firstly, most of the included trials in this study were completed in developed countries, and they fail to reflect the actual characteristics of the broader sample. In addition, few trials completed the registration process on the relevant trial platforms, which may directly affect the stability of our evidence. Finally, although we used quantitative analyses to ensure the accuracy of the effects of planning interventions in promoting PA, the sources of moderate heterogeneity observed (e.g., different planning intervention strategies, intervention delivery models, samples, and sample size selection, etc.) need to be further explored. # Conclusions This review identifies that planning interventions are effective in improving PA behavior among the general population. In addition, the results of this review provide sufficient evidence that the effects of planning interventions vary according to different moderators and contexts. As effective intervention strategies with low cost, planning intervention should be broadly promoted and applied by health practitioners and policymakers. Supplementary Materials: The following materials are available online at https://www.mdpi.com/ article/10.3390/ijerph19127337/s1, PRISMA 2020 checklist, search strategy, [fig_ref] Figure 1: Flow chart of study design by PRISMA 2020 [/fig_ref] : funnel of plot, [fig_ref] Figure 2: Risk of bias graph. [/fig_ref] : Egger plot. Author Contributions: Conceptualization, S.P. and F.Y.; methodology, S.P. and J.L.; formal analysis, S.P.; investigation, S.P. and F.Y.; writing-original draft preparation, S.P.; writing-review and editing, S.P. and J.L.; supervision, A.T.O.; project administration, S.P.; funding acquisition, S.P. All authors have read and agreed to the published version of the manuscript. [fig] Figure 1: Flow chart of study design by PRISMA 2020. [/fig] [fig] Figure 2: Risk of bias graph. [/fig] [fig] Figure 3: Risk of bias summary for included studies. [/fig] [fig] Funding: This research was supported by the Fundamental Research Funds for the Central Universities (Hohai University, B200202203). Institutional Review Board Statement: Not applicable. Informed Consent Statement: Not applicable. Data Availability Statement: Data generated or analyzed during this study are included in this published article or in the data repositories listed in the references. [/fig] [table] Table 1: Characteristics of studies.Table 1. Cont. Table 1. Cont.Notes: AP: action planning; Baecke-HPA: Baecke Questionnaire of Habitual Physical Activity; BTDPAR: Bouchard Three-Day Physical Activity Record; CG: controlled group; CP: coping planning; GSLTPAQ: Godin-Shephard Leisure-Time Physical Activity Questionnaire. IG: intervention group; IPAQ: International Physical Activity Questionnaire; KPAS: Kaiser Physical Activity Survey; LTEQ: Leisure Time-Exercise Questionnaire; LTPA: leisure-time physical activity; M: month; METs: metabolic equivalents; MVPA: moderate-vigorous physical activity; PARA-SCI: Physical Activity Recall Assessment for Individuals with Spinal Cord Injury patients; PASE: Physical Activity Scale for the Elderly; PRISCUS-PAQ: PRISCUS-Physical Activity Questionnaire; SQUASH: Dutch Short Questionnaire to Assess Health Enhancing Physical Activity; SWET: self-report walking and exercise tables; W: week. [/table] [table] Table 2: Primary results and subgroup analyses. [/table]

Idiopathic Granulomatous Hypophysitis Mimicking Pituitary Abscess Idiopathic granulomatous hypophysitis (IGH) is a rare inflammatory disease of the pituitary that commonly presents with enlargement of the pituitary gland. Clinically and radiologically, IGH is a rare sellar entity easily to be misdiagnosed as a pituitary adenoma.Through such a case, we aim to present this rarity and emphasize the importance to correctly diagnose confusing pituitary lesions comprehensively by clinical presentations, radiological signs, and biopsy.We present an uncommon case of IGH in a 19-year-old man. The patient was admitted to the hospital with severe headache, vomiting, and vision's sharp decline. Magnetic resonance imaging showed a sellar lesion with obvious cystic change and ring enhancement. The disease course including diagnosis and treatment was presented and analyzed in detail. The pertinent literature is reviewed regarding this uncommon entity.The patient underwent surgical exploration and partial resection via the transsphenoidal approach. The pathologic findings suggested IGH giving no significant evidences of systemic granulomatous disease and venereal disease. Large dose methylprednisolone was then used. The pituitary function recovered, but there was no apparent improvement of his vision.IGH is a rarely occurred inflammatory disease of unknown etiology. It is difficult to diagnose preoperatively and is often misdiagnosed. Although rare, IGH should be kept in mind in terms of differential diagnosis of sellar region lesions.(Medicine 94(28):e1099)Abbreviations: CSF = cerebrospinal fluid, GH = granulomatous hypophysitis, IGH = idiopathic granulomatous hypophysitis, LH = lymphocytic hypophysitis, MRI = magnetic resonance imaging, PRL = prolactin, VA = visual acuity, VF = visual field, WBC = white blood cell. ISSN: 0025-7974 # Introduction H ypophysitis is relatively a rare disorder, with an estimated incidence of 1 case per 9 million people per year and imaging diagnosis is difficult. [bib_ref] Lymphocytic and granulocytic hypophysitis: a single centre experience, Buxton [/bib_ref] They encompass a wide spectrum of pathology including lymphocytic hypophysitis (LH), granulomatous hypophysitis (GH), local manifestations of systemic disease, and a multitude of infectious processes. [bib_ref] Primary (autoimmune) hypophysitis: a single centre experience, Khare [/bib_ref] GH is a chronic inflammatory condition of the pituitary, first described by Simmonds in 1917. [bib_ref] Granulomatous hypophysitis, Bhardwaj [/bib_ref] Majority of pituitary granulomas represent a specific lesion such as syphilis, tuberculosis, sarcoidosis, or histiocytosis-X. In absence of any demonstrable causative agent, the process is termed idiopathic granulomatous hypophysitis (IGH). The most common presenting symptom was headache, followed by chronically progressive visual changes. Magnetic resonance imaging (MRI)-specific data for IGH was poorly reported, with pituitary enlargement the most common feature. Contrast enhancement and pituitary stalk thickening may also appear. Clinically and radiologically, IGH is a rare sellar entity easily to be misdiagnosed as a pituitary adenoma. Herein, we present a case of a 19-year-old man suffering from IGH complaining of vision's sharp decline to blindness within 1 week. Uniquely, the MRI showed a sellar lesion with obvious cystic change and ring enhancement, very much mimicking a pituitary or peripituitary abscess. To the best of our knowledge, this is the first reported case of IGH presenting in this manner giving his unique clinical and image features. Because the rarity of IGH often leads to the misdiagnosis of either another inflammatory and infectious disease or a pituitary tumor, we also review the latest literature on the etiology, diagnosis, and treatment of IGH. ## Case report A 19-year-old Chinese man, without particular previous medical history, had complained of a bilateral temporal headache for more than half a year. The pain was diffuse, dull, and paroxysmal, and could be alleviated by rest. He also presented with transient fever (maximum 39.28C) for several times. He did not seek medical services until nausea and vomiting occasionally occurred especially in the morning 1 month after the onset. He underwent a head computed tomography scan at the local hospital, showing a 15 mm  10 mm cyst in the pineal region (just a chance finding). No apparent changes were found in other intracranial structures including the sella turcica area. He was treated pharmacologically with analgesics. However, his condition did not improve. After 2 weeks, because of frequent nausea and vomiting, he came to our hospital. The patient was down in spirits with heavy complexion. In addition to the above symptoms, he also complained a mildly decreased bilateral visual acuity (VA) in recent weeks. He denied polyuria, polydipsia, and sexual hypoactivity, and had no symptoms of unconsciousness, convulsion, epilepsy, and cognitive disorder, and no special circumstances regarding his family history or personal history related to his presentation were identified. Neurological examination showed a normal function of both sensation and motor at 4 limbs. No apparent changes in superficial and deep tendon reflexes were observed. Both superficial and deep tendon reflexes were normal. Pathological signs and ataxia were absent. Upon rough ophthalmologic examination, his left palpebral fissure was relatively small with ptosis, and pupillary light reflex was insensitive on the right side. The left VA was 0.8 and the right 0.6. There was suspicious defect in right upper quadrant of his right eye. However, his VA declined sharply. Just within 3 days while waiting for his head MRI scanning, the patient complained he even could not see anything with his right eye, and his vision on the left side blurred further. Upon specialized ophthalmologic examination, relative afferent pupillary defect on the right side was strong positive and fundoscopic examination was unremarkable. Temporal incomplete hemianopia and nasal scotomas of his left eye were observed [fig_ref] FIGURE 1: Visual field test of the left eye showed temporal incomplete hemianopia [/fig_ref]. His right VA was too low to undergo a visual field (VF) test. Head MRI revealed a nodular lesion (12 mm  10 mm  8 mm) at the right part of suprasellar region in the anterior of optic chiasma. The lesion's interior was cystic, which was isointensity as gray matter on T1-weighted and T2weighted images. After injection of an intravenous contrast agent, the lesion was obviously ring-enhanced. The lesion circumvented the right internal carotid artery (C7 segment) and had no clear limit with visual chiama. In addition, the pituitary stalk, funnel, right optic nerve, the back of the right gyrus rectus, and substantia perforata anterior were also involved [fig_ref] FIGURE 2: Radiological evaluation of the brain before surgery [/fig_ref]. These images were representative of the features of a pituitary or peripituitary abscess. Lumbar puncture The patient's vision continued to deteriorate. On the 6th day after his admission, he could only see his hand move with his right eye, and his left eye could only see light from the outside margin of the eyelid. He underwent surgical exploration via the transsphenoidal approach. Only partial resection was achieved. The pathologic findings revealed granulomatous changes with multinucleated giant cells and macrophages infiltration [fig_ref] FIGURE 3: Histopathologic test [/fig_ref]. Once the histopathologic diagnosis was clear and definite, the patient was further undergone cutaneous, skeletal, visceral, and laboratory examinations for systemic granulomatous disease such as tuberculosis, syphilis, sarcoidosis, brucellosis, and histiocytosis-X. Owing to no significant evidences of systemic granulomatous disease and venereal disease were found, the patient was finally diagnosed with IGH. Giving the resection was limited, impulsion therapy with large dose methylprednisolone was then used after the final diagnosis. The patient's headache and vomiting have been completely cured, and his Pituitary function is primarily normal after surgery, but there is no apparent improvement of his vision. He is now under outpatient follow-up care. # Discussion Inflammatory diseases involving the pituitary gland are rare when compared with adenomas. Histologically, inflammatory hypophysitis is classified into 5 subtypes: lymphocytic, granulamatous, xanthomatous, xanthogranulomatous, and necrotizing. [bib_ref] Primary nonnecrotizing granulomatous hypophysitis mimicking pituitary adenomas, Yildirim [/bib_ref] [bib_ref] Autoimmune hypophysitis: new developments, Takahashi [/bib_ref] Hypophysitis can also be classified as idiopathic hypophysitis and secondary hypophysitis which may be caused by systemic inflammatory disorders such as tuberculosis, Wegener's granulomatosis, histiocytosis-X, and sarcoidosis. [bib_ref] Primary nonnecrotizing granulomatous hypophysitis mimicking pituitary adenomas, Yildirim [/bib_ref] [bib_ref] Distinct radiological and clinical appearance of lymphocytic hypophysitis, Unluhizarci [/bib_ref] The majority of inflammatory pituitary lesions occur in women, are related with the postpartum period, and most of them are in the form of LH. [bib_ref] Granulomatous hypophysitis: presentation and MRI appearance, Unlu [/bib_ref] IGH, an extremely rare chronic pituitary inflammation, has been reported in <1% of sellar lesions based on surgical findings with the transsphenoidal approach. [bib_ref] Idiopathic granulomatous hypophysitis: comments and a disease registry, Hunn [/bib_ref] Although no definite etiology has been described, there are differing perspectives on the pathogenesis of IGH. The mainstream view that the pathogenesis of IGH was thought to be attributed to autoimmunity. [bib_ref] Idiopathic granulomatous hypophysitis: comments and a disease registry, Hunn [/bib_ref] However, in our case, no autoimmune background was found. The relationship, if any, between IGH and LH is unclear. 9,10 Some authors feel these conditions are different or are opposite ends of the spectrum of same disease, with fibrosis representing the end stage of the inflammatory process. [bib_ref] Autoimmune hypophysitis: new developments, Takahashi [/bib_ref] Others suggest IGH differs from LH in certain epidemiological features, which are mostly associated with pregnancy and autoimmune diseases. A recent systematic review of 82 cases of IGH demonstrated a female predilection of IGH, and showed that most cases of IGH exhibit lymphocytic infiltration of the pituitary gland. In addition, the mean age of presentation in IGH is 43 years, some 8 years subsequent to the mean age of presentation in LH, suggesting the former view mentioned above. [bib_ref] Idiopathic granulomatous hypophysitis: a systematic review of 82 cases in the literature, Hunn [/bib_ref] Headaches are the most common presenting symptom of GH. [bib_ref] Idiopathic granulomatous hypophysitis: a systematic review of 82 cases in the literature, Hunn [/bib_ref] Patients may also present with chronically progressive chiasmal compression, hypopituitarism, amenorrhea-galactorrhea, hyperprolactinemia, fatigue, and diabetes insipidus. [bib_ref] Diagnosis and classification of autoimmune hypophysitis, Falorni [/bib_ref] In our case, the patient demonstrated a sudden onset of severe blurred vision and VF's sharply increased defect, which is different from previous reports. On MRI, there was no clear limit between the IGH lesion and the thickening visual chiama and right optic nerve. Vision's sharp decline suggested the lesion developed rapidly and furiously invaded the visual pathway. In addition, lateral expansion of pituitary mass into the cavernous sinus could compress III, IV, or VI cranial nerves, resulting in diplopia and subsequently ocular misalignment. The patient's ptosis was mostly secondary to oculomotor paralysis because of pressure and stimulation. Given this, we once considered a possible diagnosis of a rapidly developing optic glioma, for optic glioma typically shows partial or total loss of vision or changes in the optic nerves and nausea and vomiting may also be present. [bib_ref] Visual function and optic pathway glioma: a critical response, Gutmann [/bib_ref] On MRI, it shows as an enlargement, kinking, and buckling of the optic nerve, and may also appear cystic degeneration. [bib_ref] Optic nerve glioma: an update, Nair [/bib_ref] However, optic nerve glioma is a rare kind of cancer, usually slow-growing and found in children. It is rarely found in individuals over the age of 20. Apart from the visual impairment, the patient also demonstrated a mild hyperprolactinaemia, which may due to the compression of the pituitary stalk or the inflmmatory process itself preventing the inhibitory regulation of PRL release by hypothalamic dopamine.Imaging appearances of different types of primary hypophysitis are very similar, and it is really difficult to differentiate hypophysitis from the commonest sellar region lesion pituitary adenoma just based on the MRI which is far from specific enough. [bib_ref] Autoimmune hypophysitis: new developments, Takahashi [/bib_ref] But in most clinical situations, as for hypophysitis, the pituitary enhancement is usually intense, homogeneous, and symmetrical, and the invasive changes of the sella turcica floor are rare. [bib_ref] Autoimmune hypophysitis, Dhanwal [/bib_ref] It is even harder to diagnose IGH preoperatively; available diagnostic clues on MRI are that the neurohypophysis' bright spot is rarely seen and pituitary stalk usually gets thicker. [bib_ref] Idiopathic granulomatous hypophysitis: a systematic review of 82 cases in the literature, Hunn [/bib_ref] Perez-Nunez 16 once described a case of LH imageologically mimicking a pituitary abscess presenting with the MRI of a sellar cystic lesion with a hypointense core surrounded by a contrastenhancing rim, contacting with, but not compressing the optic chiasma. He concluded that LH should be kept in mind when differentiate cystic ring-enhanced cystic sellar region lesions. Our case is the second reported one of primary hypophysitis and the first reported one of IGH with cystic and ringenhanced MRI appearance. It has 2 prominent features: it was located in the suprasellar region instead of saddle area and the pituitary itself was not enlarged and the cystic and ringenhanced lesion adhered tightly to the optic chiasma and nerve and resulted in a precipitously worsened vision loss. In addition, a pineal cyst was hit upon. Transsphenoidal surgery is a primary diagnostic option, especially for those whose clinical and radiological presentation is not typical and diagnosis is not confirmed. [bib_ref] Autoimmune hypophysitis, Dhanwal [/bib_ref] Histopathological findings of pituitary biopsy remain the gold standard for diagnosing primary hypophysitis. [bib_ref] Diagnosis and classification of autoimmune hypophysitis, Falorni [/bib_ref] In fact, majority of reported IGH and approximately half of the primary hypophysitis were misdiagnosed as pituitary adenomas before surgery. [bib_ref] Idiopathic granulomatous hypophysitis, Stott [/bib_ref] [bib_ref] Primary granulomatous hypophysitis: a case report and literature review, Su [/bib_ref] If primary hypophysitis is suspected, an intraoperative histology on frozen sections is recommended to confirm the diagnosis and avoid extensive surgery because hypopituitarism may occur or be worsened after extensive resection. [bib_ref] Autoimmune hypophysitis: new developments, Takahashi [/bib_ref] [bib_ref] Primary granulomatous hypophysitis: a case report and literature review, Su [/bib_ref] As the natural history of IGH is incompletely understood yet, its treatment is still controversial. [bib_ref] A case of idiopathic granulomatous hypophysitis, Chung [/bib_ref] Surgery not only provides live tissue for histological diagnosis, but can rapidly decompress the mass lesion, thereby resolving headache and visual deficits immediately. Hypopituitarism resolves after surgery in some cases, but many patients remain on full hormone replacement therapy. Satisfactory response to high-dose steroid therapy or anti-inflammatory and immunosuppressive (methotrexate, cyclosporine A, azathioprine) treatments have also been widely reported. [bib_ref] Primary granulomatous hypophysitis: a case report and literature review, Su [/bib_ref] [bib_ref] Granulomatous hypophysitis: two case reports and literature review, Shi [/bib_ref] [bib_ref] Lymphocytic hypophysitis with dacryoadenitis in a male patient: non-invasive diagnosis and high-dose..., Lu [/bib_ref] What is noteworthy is that in the present case, there was no apparent improvement of the patient's vision after recovery of normal pituitary function. We guess there are at least 3 possible reasons accounting for that. First, the resection surgery was not total but partial. Although impulsion therapy with large dose methylprednisolone was used after surgery, there might still be some left lesion tissues which could still exert mass compression effect and stimulatory effect on the optic chiasm. Second, the patient's preoperative VF and VA were too poor and deteriorated rapidly. According to a multivariate analysis for pituitary adenomas, the only independent predictor of postoperative recovery of VF was the degree of preoperative VF deficit. Postoperative VF recovery was also related to both the duration of symptoms and the severity of preoperative VA. Third, the pituitary lesion nature in this case is IGH rather than the commonest adenoma, whose pathogenic effect may be not limited to simple compression. So in this circumstance, the optic chiasm could encounter some nonreversible pathologic change such as optic atrophy. [bib_ref] The time course of visual field recovery following transphenoidal surgery for pituitary..., Gnanalingham [/bib_ref] All in all, VF recovery may be related to many factors including differences in the patient population studied, differences in preoperative deficits in VF and VA, the lesions' pathological nature, the extent of lesion resection, the dose of postoperative radiotherapy, and so on. # Conclusion We report an uncommon case of suprasellar IGH with cystic and ring-enhanced MRI appearance resulting in a precipitously worsened vision loss. IGH is a rarely occurred inflammatory disease of unknown etiology. It is difficult to diagnose preoperatively and is often misdiagnosed. With this article, we not only aim to present this rare entity, but also to emphasize the importance to correctly diagnose cystic ring-enhancing sellar lesions to ensure the patient receives proper treatment. [fig] FIGURE 1: Visual field test of the left eye showed temporal incomplete hemianopia (black areas) and nasal scotomas (crimson areas). [/fig] [fig] FIGURE 2: Radiological evaluation of the brain before surgery. T1-weighted axial gadolinium-enhanced MRI demonstrated a ringenhancing cystic lesion at the right part of suprasellar region in the anterior of optic chiasma (arrows) (A, C, D). T2-weighted image demonstrates the same lesion as in the previous image (arrow) (B). A cyst in the pineal region was also showed with long-T1 and long-T2 signals (arrows) (E, F). [/fig] [fig] FIGURE 3: Histopathologic test (hematoxylin-eosin staining, 200Â) revealed granulomatous changes with multinucleated giant cells and macrophages infiltration (on the bottom left of the field area). On the right was the surrounding adenohypophysial tissue. [/fig]

Flexible All-organic, All-solution Processed Thin Film Transistor Array with Ultrashort Channel Shrinking the device dimension has long been the pursuit of the semiconductor industry to increase the device density and operation speed. In the application of thin film transistors (TFTs), all-organic TFT arrays made by all-solution process are desired for low cost and flexible electronics. One of the greatest challenges is how to achieve ultrashort channel through a cost-effective method. In our study, ultrashort-channel devices are demonstrated by direct inkjet printing conducting polymer as source/ drain and gate electrodes without any complicated substrate's pre-patterning process. By modifying the substrate's wettability, the conducting polymer's contact line is pinned during drying process which makes the channel length well-controlled. An organic TFT array of 200 devices with 2 μm channel length is fabricated on flexible substrate through all-solution process. The simple and scalable process to fabricate high resolution organic transistor array offers a low cost approach in the development of flexible and wearable electronics.Organic thin-film transistors (OTFTs) have attracted great attention for a wide range of applications, such as flexible displays 1,2 , sensors 3 , radio frequency identification tags 4 , low-cost memories 5 , and microprocessors 6 , due to their unique properties of excellent mechanical flexibility and process advantages of being compatible with high throughput and low-budget printing processes 7 . Over the years, significant efforts have been made to develop high carrier mobility, chemically and physically stable organic semiconductor materials with the reported device performance of OTFTs already better than that of amorphous silicon devices 8-11 . However, the fabrication of OTFT devices generally requires thermal evaporation through masks to deposit metal electrodes and the semiconducting layer in vacuum 1-14 . The process is known to be costly and difficult to scale up. To fully exploit the potentials of organic materials in the applications of low cost and flexible electronics, all-organic TFTs made by all-solution process are desired 15 .Various techniques have been developed to fabricate all-organic TFTs. Conventional photolithography, which is the standard technique in inorganic semiconductor industry, has been explored for patterning polymer electrodes in bottom contact OTFTs. For example, Halik et al. fabricated all-organic pentacene TFTs on flexible polymeric substrates with photolithography-patterned PEDOT:PSS contacts 16 . A similar photo-induced patterning approach, named photochemical patterning, was developed by Gelinck et al. They demonstrated fully-solution-processed and all-polymer integrated circuits with conductive polyaniline electrodes defined by photochemical conversion through a shadow mask 17 . Although photolithography is a mature process and capable of high resolution patterning, its application in patterning OTFTs is rather limited due to degradation of organic semiconductors upon exposure of the etching chemicals.Screen printing, which is suitable for large scale production and much cheaper than photolithography, has been exploited in OTFTs much earlier than photolithography. Garnier et al. demonstrated the first all-organic TFTs back in 1994 of which the source, drain and gate electrodes were all screen-printed from a graphite based ink, while the small molecular semiconductor was thermally evaporated 18 . Most recently, Hyun et al. adopted a photolithography-patterned silicon sheet as the stencil to improve the resolution of screen printing 19 . All-solution processed, electrolyte gated transistors were fabricated with screen-printed graphene source and drain (S/D), and an aerosol-jet printed poly(3,4-ethylenedioxythiophene) polystyrene sulfonate (PEDOT:PSS) gate. However, the obtained channel length of 90 μm is not narrow enough to meet the demand for large drive current and high switching speed. Scientific RepoRts | 6:29055 | DOI: [bib_ref] Influence of Alkyl Chain Branching Positions on the Hole Mobilities of Polymer..., Lei [/bib_ref].1038/srep29055 Microcontact printing, which transfers patterns from stamps, can achieve a resolution as high as that of photolithography while retaining low cost. Parashkov et al. employed the process to pattern an electron-transfer blocking layer on a gold plate to realize selective electro-polymerization of PEDOT:PSS S/D 20 . The electrodes were then transferred onto a polyimide substrate where pentacene and polyvinyl alcohol (PVA) were deposited as the semiconductor and dielectric layer. The PEDOT:PSS gate was screen printed. The final channel length is as small as 10 μm. However, the costly metal deposition in vacuum for preparing the electro-polymerization template was required to deposit the electrodes. Another simple and cost-effective patterning technique is inkjet printing. The advantages of its fully additive feature, flexibility in pattern design, and low material consumption, make inkjet printing an attractive deposition technique in OTFT fabrication. The patterning resolution of inkjet printing, however, is generally tens of micrometers owing to the difficulty in controlling the droplet size and ink-spreading on substrates. To overcome the resolution limitation, Sirringhaus et al. demonstrated an approach by depositing the functional ink onto a substrate containing a predefined surface-energy pattern that was able to split the deposited ink droplets to form a narrow gap [bib_ref] High-Resolution Inkjet Printing of All-Polymer Transistor Circuits, Sirringhaus [/bib_ref] [bib_ref] Dewetting of conducting polymer inkjet droplets on patterned surfaces, Wang [/bib_ref]. Sub-micrometer channel length was achieved by inkjet printing PEDOT:PSS ink. The process requires photolithography or electron-beam lithography to prepare the hydrophobic strips. To further exploit the direct-writing capability by inkjet printing, the authors developed a lithography-free nanopatterning technique in which a self-assembled surface layer induced the dewetting of PEDOT:PSS resulting in sub-micrometer size channels [bib_ref] Self-Aligned Inkjet Printing with Sub-Hundred-Nanometer Resolution, Sele [/bib_ref]. However, the additional surface layer sometimes causes defects at electrode contacts, and the dewetting process is time consuming. All aforementioned techniques require multi-step procedures to pattern organic electrodes in order to achieve the narrow channel length. In our contribution, we develop a direct-writing technique to pattern organic electrodes with high resolution by inkjet printing. All-organic and all-solution processed TFT arrays were fabricated on a flexible substrate. By modifying the surface wettability of the polyethylene terephthalate (PET) surface with PVA, a channel length around 2 μm defined by PEDOT:PSS electrodes was achieved. To the best of our knowledge, this is the highest resolution ever reported for directly inkjet-printed electrodes without any complicated substrate pre-patterning process. A 10 by 20 OTFT array were fabricated across a 3 cm × 3 cm flexible substrate showing good uniformity and high yield. Our study demonstrates that by carefully controlling ink spreading on substrates, high resolution OTFTs are achievable by direct inkjet printing. # Results To fabricate OTFTs on a flexible substrate, a 200 μm thick PET foil first went through 20 mins oxygen-plasma treatment to increase the surface energy. PVA in deionized water (20 mg/mL) was spin-coated onto PET film to form a 50 nm buffer layer to modify the PET surface. On top of the PVA surface modification layer, S/D electrodes were fabricated by line printing conducting polymer PEDOT:PSS [bib_ref] Line printing solution-processable small molecules with uniform surface profile via ink-jet printer, Liu [/bib_ref]. The organic semiconductor layer was deposited by spin-coating poly [3,6-bis(40-dodecyl [bib_ref] Design and realization of a flexible QQVGA AMOLED display with organic TFTs, Steudel [/bib_ref] [bib_ref] Microcontact Printing as a Versatile Tool for Patterning Organic Field-Effect Transistors, Parashkov [/bib_ref] bithiophenyl-5-yl)-2,5-bis(2-hexyldecyl)-2,5-dihydropyrrolo pyrrole-1,4-dione] (PDQT) in chloroform (6 mg/mL). The thickness of the active layer is about 60 nm. On top of the active layer, a 500 nm film of polymethylmethacrylate (PMMA) was spin-coated from its n-butyl acetate solution (50 mg/mL) as the gate dielectric layer. An additional ultrathin polyethylene glycol (PEO) layer was spin-coated from a 5 mg/mL solution in methanol to modify the PMMA surface to enhance its wettability for the subsequent PEDOT:PSS gate printing, which was laid perpendicular to the S/D lines. The top-gate OTFT structure is schematically illustrated in [fig_ref] Figure 1: OTFT structure [/fig_ref]. All the processes are solution-based, and all the processes are carried out in air at room temperature. Reducing the channel length to achieve high device integration density, high operation speed and low power consumption is critical in OTFT development. Grau et al. reported gravure-printed OTFTs with 5 μm channel length [bib_ref] Fully High-Speed Gravure Printed, Low-Variability, High-Performance Organic Polymer Transistors with Sub-5 V..., Grau [/bib_ref]. Yoshimura et al. achieved 4 μm channel length by inkjet printing silver inks [bib_ref] High-speed operation in printed organic inverter circuits with short channel length, Yoshimura [/bib_ref]. Fukuda et al., taking advantage of the high-resolution off-set printing and the their customized silver ink, reached a submicrometer channel length of 0.6 μm 26 . However, the studies to achieve narrow channel length all used metal inks. Though direct inkjet-printing conducting polymers (mainly PEDOT:PSS) as S/D electrodes in OTFT devices without pre-patterning substrate has been demonstrated 27 , the typical channel length is between 20 to 50 μm owing to the ink spreading on substrates and droplet positioning error [bib_ref] High-Resolution Inkjet Printing of All-Polymer Transistor Circuits, Sirringhaus [/bib_ref]. To overcome the problem, we propose here that by modifying the surface energy of the substrate with a hydrophilic and dissolvable layer, namely, the PVA buffer layer, accurate PEDOT:PSS patterns of short channels becomes possible due to the contact line pinning effect and excellent film morphology. To obtain ultrashort channel, the S/D electrodes' patterning process is crucial. During the inkjet printing process, upon striking the substrate, the PEDOT:PSS droplet spreads first as driven by the kinetic energy. Afterwards, it shrinks because of the surface tension, and finally dries to form a stable shape [bib_ref] Effects of droplet size and solute concentration on drying process of polymer..., Fukai [/bib_ref]. After inkjet printing PEDOT:PSS in the polar solvent (water) on a non-polar PET surface, due to the high contact angle of 107°, the consecutively printed PEDOT:PSS droplets can't form a continuous line. Instead, isolated islands were formed, as demonstrated in [fig_ref] Figure 2: Printed PEDOT [/fig_ref]. To increase the hydrophilicity of the substrate, PVA was spin-coated to modify the PET surface. The atomic force microscopy (AFM) image in [fig_ref] Figure 2: Printed PEDOT [/fig_ref] shows the PVA has a very smooth surface with a root mean square (RMS) roughness of about 1.2 nm. On PVA, the contact angle of PEDOT:PSS was reduced to 38°. As the result, a uniform electrode line was formed by the consecutively printed PEDOT:PSS droplets as shown in [fig_ref] Figure 2: Printed PEDOT [/fig_ref]. The three-dimensional picture from the white light interference microscope shows a relative smooth surface of the PEDOT:PSS line free of the coffee ring [fig_ref] Figure 2: Printed PEDOT [/fig_ref]. It is determined by the cross-section profile that the line width is about 106 μm, and the line thickness is about 100 nm. A common practice to increase the substrate surface energy is UV/ozone or oxygen plasma treatment [bib_ref] Fully-printed high-performance organic thin-film transistors and circuitry on one-micron-thick polymer films, Fukuda [/bib_ref] [bib_ref] Plasma Treatment for Improved Bonding: A Review, Liston [/bib_ref]. However, the wettability provided by the treatments is not stable in air [bib_ref] Surface modification of indium-tin-oxide anode by oxygen plasma for organic electroluminescent devices, You [/bib_ref] [bib_ref] Oxygen and nitrogen plasma hydrophilization and hydrophobic recovery of polymers, Jokinen [/bib_ref]. With PVA film, the hydrophilic layer guarantees the uniform coating of the PEDOT:PSS electrode array across a large area during the printing process without any wettability deterioration. Moreover, the PVA layer can pin the contact line during PEDOT:PSS film formation. [fig_ref] Figure 2: Printed PEDOT [/fig_ref] shows the drying process of the PEDOT:PSS droplets on PVA/PET substrate. The total drying time t f was 600 s. The contact line (as denoted by the dash lines) was strongly pinned due to the dissolution of PVA underneath the PEDOT:PSS droplet, and the contact line did not withdraw during the whole drying process as shown at t = 60 s, 180 s, 240 s, 420 s , 540 s, and 600 s, respectively. In our early work, PEDOT:PSS was inkjet printed on an oxygen-plasma treated PMMA surface [bib_ref] High-performance, all-solution-processed organic nanowire transistor arrays with inkjet-printing patterned electrodes, Liu [/bib_ref]. The contact line withdrew during PEDOT:PSS solvent evaporation due to insufficient interfacial adhesion. The minimum channel length of 20 μm was determined by the receding radius of the droplet. In the current study, with surface modification of the hydrophilic PVA, precise control of the line gap and line width can be realized owing to the pinned contact line [bib_ref] Contact-Line Recession Leaving a Macroscopic Polymer Film in the Drying Droplets of, Kajiya [/bib_ref]. As the result, an ultrashort channel was achieved by direct inkjet printing through careful calculation and adjustment of the printing parameters without any complicated substrate pre-patterning process. The stable line width through inkjet printing can be estimated by the following equation (1) where d 0 is the droplet diameter in air, θ is the contact angle of the droplet on the substrate, and p is the distance between the consecutive droplets' landing positions. It's clear that for a given printer cartridge with the fixed d 0 and θ, p is the only parameter which can be optimized. To obtain fine lines with smooth edges, p can neither be too small nor too large [bib_ref] Inkjet-Printed Line Morphologies and Temperature Control of the Coffee Ring Effect, Soltman [/bib_ref]. The optimized p for PEDOT:PSS jetted from a 30 μm nozzle, is around 50 μm, which resulted in a line width of about 106 μm without coffee ring [fig_ref] Figure 2: Printed PEDOT [/fig_ref]. Thus, by carefully adjusting the line-center distance between two PEDOT:PSS electrodes, the printed channel length can be determined. By setting the printing line-center distance at 118 μm after taking into account the line width and the substrate positioning accuracy, a 2 μm channel length was successfully achieved. To test the printing uniformity, 200 structures were fabricated on a 3 cm × 3 cm PET substrate with 2 μm channel length and 970 μm channel width. The microscopic pictures and AFM topography of the printed channels are shown in [fig_ref] Figure 3: A printed array of ultrashort channels [/fig_ref]. The inset of [fig_ref] Figure 3: A printed array of ultrashort channels [/fig_ref] shows that the all-solution processed OTFTs are flexible and transparent. The channel length distribution in [fig_ref] Figure 3: A printed array of ultrashort channels [/fig_ref] shows the direct inkjet printing is reliable and repeatable (2.0 μm in average with 0.5 μm standard deviation). No shortening of the S/D electrodes was detected confirming the high yield of the device array fabrication. The typical transfer characteristics of the 2 μm channel OTFT device is shown in [fig_ref] Figure 4: Performance of OTFTs [/fig_ref]. The maximum hole mobility is (0.64 ± 0.27) × 10 −3 cm 2 /V · s, ON/OFF ratio is (0.92 ± 0.48) × 10 3 , and turn-on voltage is 1.94 ± 0.32 V. The statistics is calculated from a total of 96 devices. For comparison, OTFTs with thermal-evaporated Au S/D electrodes and a channel length of 70 μm (W/L = 4) were fabricated. As shown in [fig_ref] Figure 4: Performance of OTFTs [/fig_ref] , the devices exhibit a mobility of 0.55 cm 2 /V·s, an ON/OFF ratio of 3.26 × 10 4 , and a turn-on voltage at −5.5 V. A previous study on non-annealed PDQT devices using a bottom-gate top-contact structure with evaporated Au electrodes reported a mobility of 0.89 cm 2 /V·s, an ON/OFF ratio of 10 7 , and a threshold voltage of −2.6 V 37 . The performances of the Au devices indicate that the poor performance of the 2-μm-channel all-organic devices is not due to the semiconducting or the dielectric layer, but the printed PEDOT:PSS electrodes and their interface to the semiconducting layer. It is observed that while the OFF current of 2-μm-channel devices remains at the same level as the Au device, the ON current is about an order of magnitude smaller, which directly reduces the ON/OFF ratio to about 10 3 . It is also noticed that the slope of the square root of drain current quickly decreases as the gate voltage increases. Therefore, it is speculated that the poor performance may be the result of the relatively large contact resistance at the printed electrodes. To confirm it, all-solution processed, all-organic TFTs with channel lengths of 30 μm, 20 μm and 10 μm were fabricated. The channel width was kept at 970 μm. Transfer characteristics are presented in [fig_ref] Figure 4: Performance of OTFTs [/fig_ref] ,c gives the statistical distribution of the mobility and ON/OFF ratio for the devices with different channel length. The statistics are calculated from a total of 46, 93, and 61 devices, for 30 μm, 20 μm, and 10 μm channel length, respectively. The device performances are summarized in . The mobility shows a decreasing trend as the channel length shortens [fig_ref] Figure 4: Performance of OTFTs [/fig_ref] , which is consistent with the effect of large contact resistance in TFTs. By further plotting the channel resistance (normalized to the channel width) as a function of channel length in [fig_ref] Figure 4: Performance of OTFTs [/fig_ref] (at the gate voltage of −5 V), a contact resistance of about 15.0 MΩ · cm is identified. The value is about two to three orders of magnitude larger than typical solution-processed OTFTs. Such large contact resistance will limit the drain current at high gate voltages. As a result, calculating mobility from typical equations will lead to much smaller values. The origin of the large contact resistance could come from the relatively large injection barrier from PEDOT:PSS electrodes to the semiconducting PDQT, as well as the large bulk resistance of intrinsic PEDOT:PSS [bib_ref] High-Resolution Inkjet Printing of All-Polymer Transistor Circuits, Sirringhaus [/bib_ref]. Further device optimization by, for example, inserting an injection layer between PEDOT:PSS electrodes and the PQDT layer, or raising the conductivity of PEDOT:PSS, will improve the device performance [bib_ref] Contact engineering in organic field-effect transistors, Liu [/bib_ref] [bib_ref] Highly Conductive PEDOT:PSS Nanofibrils Induced by Solution-Processed Crystallization, Kim [/bib_ref]. . Summary of the OTFT performance at different channel length. In summary, all-organic and all-solution processed OTFT array is fabricated on flexible substrate PET. The source, drain and gate electrodes are deposited by inkjet printing conducting polymer PEDOT:PSS. By modifying the PET substrate with water-dissolvable PVA, the contact line of PEDOT:PSS is pinned during the drying process resulting in well-defined edges. The pinning effect subsequently enables precise control of the printed channel length. Ultrashort channel of 2 μm is successfully realized without any complicated substrate's pre-patterning process. No short circuiting was found by printing a large array with 200 electrode pairs. Due to the large contact resistance, the printed devices show inferior performances to those with evaporated metal electrodes. By further reducing the linewidth of the printed patterns as well as optimizing the device performance, application of all-printed, high resolution TFTs in flexible displays, sensor chips, and radio-frequency identification tags will become practical. # Methods Materials. All the chemicals and materials were purchased and used as received unless otherwise noted. PEDOT:PSS Clevios PH 500 was purchased from Heraeus Deutschland GmbH & Co. KG. PVA (98-99% hydrolyzed) was purchased from Alfa Aesar. To make PVA solution (20 mg/mL) in deionized water, the solution was heated to 90 °C for 5 hours. PDQT (molecular weight > 30,000) was purchased from Lumtec Corp. PMMA, PEO, and all the solvents were purchased from Aldrich, Inc. All the solutions were filtered through a 0.45 μm filter before use. Device Fabrication and Characterization. PEDOT:PSS printing was carried out on an inkjet printer (Jetlab II) from Microfab Technologies, Inc. The printer head's orifice diameter is 30 μm. Film thickness and surface morphologies were obtained from a Dektak 150 surface profiler (Veeco Instruments, Inc.). The three-dimensional white light interference microscopy pictures were recorded by Veeco Instruments' NT 9300 surface profiler. The optical microscopic images were taken with a Nikon Eclipse E600 POL with a DXM1200F digital camera. The transistor characteristics of the devices were measured using an Agilent 4155C semiconductor parameter analyzer connected to a Cascade manual probe station. [fig] Figure 1: OTFT structure. (a) Molecular structures of PEDOT:PSS and PDQT. (b) Device structure of all-organic, all-solution processed OTFTs with inkjet-printed PEDOT:PSS electrodes on flexible substrate. [/fig] [fig] Figure 2: Printed PEDOT:PSS lines on the PVA modified substrate. (a) The contact angle of PEDOT:PSS on PET surface is 107°. Line printing on the substrate results in isolated islands due to the small solid surface energy. After PVA modification, PEDOT:PSS contact angle becomes 38°. A continuous line is formed by line printing PEDOT:PSS. (b) The AFM picture shows the smooth PVA-modified PET surface. (c) The three-dimensional white light interference microscopic picture and the cross-sectional profile of the printed PEODT:PSS line on PVA-modified PET. (d) Sequential picture of PEDOT:PSS drying process on PVA-modified PET. t f is the total drying time. [/fig] [fig] Figure 3: A printed array of ultrashort channels. (a) The printed PEDOT:PSS array (10 × 20) on the flexible PET substrate with 2 μm channel length. Inset: Picture of the array on the 3 cm × 3 cm substrate, bended with bending radius of about 5 mm. (b) The zoom-in picture of the array. (c) Picture of a single channel. (d) AFM picture of the PEDOT:PSS lines with a gap of about 2 μm. (e) The channel-length distribution of the 200 channels. [/fig] [fig] Figure 4: Performance of OTFTs. (a) Transfer characteristics of the devices with printed PEDOT:PSS electrodes. The channel width is fixed at 970 μm, and channel lengths are 2 μm, 10 μm, 20 μm and 30 μm, respectively. (b) Transfer characteristic of the device with evaporated Au S/D electrodes. W/L = 280 μm/70 μm. (c) The dependence of mobility and ON/OFF ratio on the cannel length. (d) The channel resistance of devices with different channel lengths (normalized to the channel width). The intercept at the y axis indicates the contact resistance. Channel length (μm) Mobility (cm 2 /V s) ON/OFF V turn-on (V) [/fig]

Identification and expression of functionally conserved circadian clock genes in lichen-forming fungi Lichen-forming fungi establish stable symbioses with green algae or cyanobacteria. Many species have broad distributions, both in geographic and ecological space, making them ideal subjects to study organism-environment interactions. However, little is known about the specific mechanisms that contribute to environmental adaptation in lichen-forming fungi. The circadian clock provides a welldescribed mechanism that contributes to regional adaptation across a variety of species, including fungi. Here, we identify the putative circadian clock components in phylogenetically divergent lichen-forming fungi. The core circadian genes (frq, wc-1, wc-2, frh) are present across the Fungi, including 31 lichen-forming species, and their evolutionary trajectories mirror overall fungal evolution. Comparative analyses of the clock genes indicate conserved domain architecture among lichen-and non-lichen-forming taxa. We used RT-qPCR to examine the core circadian loop of two unrelated lichen-forming fungi, Umbilicaria pustulata (Lecanoromycetes) and Dermatocarpon miniatum (Eurotiomycetes), to determine that the putative frq gene is activated in a light-dependent manner similar to the model fungus Neurospora crassa. Together, these results demonstrate that lichenforming fungi retain functional light-responsive mechanisms, including a functioning circadian clock. Our findings provide a stepping stone into investigating the circadian clock in the lichen symbiosis, e.g. its role in adaptation, and in synchronizing the symbiotic interaction.The circadian clock is a well-described central molecular timekeeping mechanism that regulates biochemical and physiological processes within most organisms, helping them to perceive and respond to abiotic and biotic environmental cues. Circadian systems are characterized by oscillations that can be synchronized (entrained) by environmental cues (i.e. zeitgebers) such as light, heat, or nutrients 1 . The entrained circadian rhythms can persist without external cues with a period of roughly 24 h under free-running conditions and are capable of temperature compensation, wherein the circadian clock maintains stable rhythmicity across broad temperature ranges within which biochemical and physiological processes are regulated 2 .The central circadian oscillator of N. crassa is composed of the negative element frequency (frq) and its interactions with the White Collar Complex (WCC), composed of proteins encoded by the white collar-1 (wc-1) and white collar-2 (wc-2) genes. WCC activates transcription of frq, which ultimately represses its own expression by affecting the phosphorylation of WC-1 and WC-2 3,4 . Another essential component of the circadian regulator is Frequency Interacting RNA Helicase (FRH), which plays a role in regulating FRQ expression by variably protecting FRQ against ubiquitin-mediated degradation and suppressing frq expression via interaction with the WCC 5 . This central clock oscillator in turn regulates a variety of downstream transcriptional and post-translational modifications 6 . The N. crassa circadian clock also exhibits a temperature compensation mechanism, consistent with other circadian clock systems in plants and animals7,8.Circadian systems are present in nearly all organisms across the various kingdoms of life, and appear to have arisen at least three times during the evolution of life 9-11 . The adaptive value of the circadian system becomes obvious in populations that have lost circadian rhythms over the course of their evolutionary history, or that OPEN ## Scientific reports | (2022) 12:15884 | https://doi.org/10.1038/s41598-022-19646-y www.nature.com/scientificreports/ "switch off " their circadian clocks due to seasonal cues; in both cases, the circadian system becomes less rhythmic under conditions where rhythmicity does not provide adaptive information to the biological system. Independently-evolved populations of the cavefish Astyanax mexicanus show widespread disruption of circadian clock gene rhythmicity, as well as a reduction in rhythmic transcription compared to surface-dwelling populations [bib_ref] Repeated evolution of circadian clock dysregulation in cavefish populations, Mack [/bib_ref]. Activating overwintering mechanisms in trees involves an interplay between photoreceptors and the circadian clock, which is then disrupted for the duration of the winter [bib_ref] Mechanism of overwintering in trees, Arakawa [/bib_ref]. The circadian rhythms of Svalbard reindeer are attenuated during winter months [bib_ref] Circadian rhythmicity persists through the Polar night and midnight sun in Svalbard..., Arnold [/bib_ref] , similarly to that of other Arctic mammals such as the muskox Ovibos moschatus [bib_ref] Environmental conditions alter behavioural organization and rhythmicity of a large Arctic ruminant..., Van Beest [/bib_ref] and the red fox Vulpes vulpes [bib_ref] Circadian activity patterns of red foxes (Vulpes vulpes) in montane forests under..., Kämmerle [/bib_ref]. The circadian clock provides fitness and performance advantages in a variety of model systems, further supporting its role in environmental adaptation. In plants, the circadian clock aligns chemical defenses to herbivore feeding patterns, regulates drought responses, and anticipates pathogen attack (see [bib_ref] The circadian clock ticks in plant stress responses, Xu [/bib_ref] [bib_ref] The circadian clock ticks in plant stress responses, Xu [/bib_ref] for a recent review of the role of the circadian clock in plant biotic and abiotic stress responses). In fungi, the asexual reproductive patterns of Neurospora crassa have long been observed to be regulated by the circadian clock [bib_ref] Growth patterns in Neurospora: A biological clock in Neurospora, Pittendrigh [/bib_ref] [bib_ref] Circadian nature of a rhythm expressed by an invertaseless strain of Neurospora..., Sargent [/bib_ref] , while strains of Neurospora discreta with habitat-specific circadian rhythms maintain higher fitness in their respective habitats [bib_ref] Habitat-specific clock variation and its consequence on reproductive fitness, Koritala [/bib_ref].The interacting circadian clocks in symbiotic systems are a growing topic of interest [bib_ref] The metronome of symbiosis: Interactions between microbes and the host circadian clock, Heath-Heckman [/bib_ref] [bib_ref] Holobiont chronobiology: mycorrhiza may be a key to linking aboveground and underground..., Lee [/bib_ref]. Tightlyregulated circadian systems have been observed between corals and algae [bib_ref] Circadian clocks in symbiotic corals: The duet between Symbiodinium algae and their..., Sorek [/bib_ref] , as well as between the Hawaiian bobtail squid Euprymna scolopes and the bioluminescent bacteria Vibrio fischeri [bib_ref] Bacteria seize control of the clock, Kåhrström [/bib_ref]. The arbuscular mycorrhizal fungus Rhizoglomus irregularis also contains a functioning circadian clock system [bib_ref] Expression of putative circadian clock components in the arbuscular mycorrhizal fungus Rhizoglomus..., Lee [/bib_ref] , which has been hypothesized to play a role in the AMF-plant symbiosis. The lichen symbiosis is composed of a fungal partner (mycobiont) and a photosynthesizing partner, either a green alga and/or a cyanobacterium (photobiont), plus a more or less specific suite of associated prokaryotic and eukaryotic microorganisms (e.g. [bib_ref] Metagenomic data reveal diverse fungal and algal communities associated with the lichen..., Smith [/bib_ref] [bib_ref] The lichens' microbiota, still a mystery?, Grimm [/bib_ref]. The lichen lifestyle-a fungal nutritional mode that relies on the photosynthetic products of internally accommodated algal symbionts-occurs in unrelated lineages across the fungal tree of life, but is most prevalent in the Leotiomyceta (e.g. Lecanoromycetes, Eurotiomycetes, Dothideomycetes) within the Ascomycota [bib_ref] Evolution of lichens, Lumbsch [/bib_ref]. Lichens are ubiquitous in the landscape, thriving in a diverse range of habitats across nearly all ecosystems [bib_ref] Die Hard: Lichens, Grube [/bib_ref]. This ubiquity is likely related to lichens' capability to withstand extreme abiotic stresses, such as complete desiccation [bib_ref] Fungal-associated NO is involved in the regulation of oxidative stress during rehydration..., Catalá [/bib_ref] [bib_ref] Plasticity of a holobiont: Desiccation induces fasting-like metabolism within the lichen microbiota, Cernava [/bib_ref]. This ability to withstand stress may be due to variable stress response pathways relative to other sessile organisms; the lichen Endocarpon pustillum maintains active transcription of metabolismassociated genes during osmotic stress that are largely suppressed in plants and fungi [bib_ref] Comparative transcriptome analysis of the lichen-forming fungus Endocarpon pusillum elucidates its drought..., Wang [/bib_ref]. Although lichens have been acknowledged as a symbiosis of interest to explore symbiotic circadian clock systems [bib_ref] Holobiont chronobiology: mycorrhiza may be a key to linking aboveground and underground..., Lee [/bib_ref] [bib_ref] Genes controlling circadian rhythm are widely distributed in cyanobacteria, Lorne [/bib_ref] , little work has been done to elucidate the circadian clock mechanism in lichens aside from the identification of the putative frq ortholog in the lichen-forming fungus Umbilicaria pustulata [bib_ref] Circadian rhythms in fungi: Structure/function/evolution of some clock components, Brody [/bib_ref]. An important prerequisite step to further studies of the lichen circadian clock is to determine how conserved the core circadian clock and photosensory machinery is across phylogenetically unrelated lichen-forming fungi, as well as to determine whether this core machinery is functional. Here, we investigate the presence of putative circadian clock components across the Fungi, focusing on major lineages in the Ascomycota that include lichens. We performed this investigation using a two-pronged approach: a phylogenetic analysis of putative homologs of the core circadian clock genes frq, wc-1, wc-2, and frh, and functional validation of one core mechanism in the fungal circadian clock. We find that homologs of these four core fungal circadian clock genes are present in lichen-forming Lecanoromycetes, Eurotiomycetes and Dothideomycetes, and that these orthologs contain strongly conserved protein-coding sequences in the functional domains of these genes. We demonstrate the light-dependent expression of the core clock gene frq in two highly diverged lichen-forming lineages, U. pustulata and D. miniatum. Taken together, these results demonstrate that lichen mycobionts retain functional light-responsive mechanisms, including a functioning circadian clock, similar to those of non-lichen-forming filamentous fungi. # Materials and methods Identification and phylogenetic analysis of putative circadian clock genes. The four core fungal clock genes frq, wc-1, wc-2, and frh of the model fungus Neurospora crassa were used as queries to search for homologs in the genomes of the lichen-forming and non-lichen-forming fungi via reciprocal BLAST in Gen-Bank, through the Genome Portal of the Joint Genome Institute 36 and from Calchera et al. [bib_ref] Biosynthetic gene content of the 'Perfume Lichens' Evernia prunastri and Pseudevernia furfuracea, Calchera [/bib_ref] ; see . Putative clock genes recovered from a broad range of fungal taxa across the Ascomycota, Basidiomycota, and Mucoromycota were used for the phylogenetic analysis. We made sure to include lichenized (and if possible nonlichenized) representatives of most Leotiomycete lineages that include lichens (Lecanoromyctes, Eurotiomycetes, Dothideomycetes). Amino acid sequence alignments were performed using MAFFT v7.450 [bib_ref] MAFFT multiple sequence alignment software version 7: Improvements in performance and usability, Katoh [/bib_ref]. After alignment, sequences were trimmed using TrimAl v1.2 [bib_ref] trimAl: A tool for automated alignment trimming in large-scale phylogenetic analyses, Capella-Gutierrez [/bib_ref] , removing all columns with gaps in more than 20% of sequences in the alignment. Phylogenetic trees were inferred by maximum likelihood using RAxML version 8 [bib_ref] RAxML version 8: A tool for phylogenetic analysis and post-analysis of large..., Stamatakis [/bib_ref] using the GAMMA BLOSUM62 model and 1,000 bootstrap replicates. The presence of functional domains between lichen-forming and non-lichen-forming lineages was investigated using the protein domain and visualization tool DomainViz 41 . ## Study site and sample collection. lichen thalli of dermatocarpon miniatum and umbilicaria pustulata were harvested from co-localized (ca. 600 m apart) sites in the vicinity of Eppstein, in the Taunus region of Germany (50°08′N 08°24′E). Single thallus fragments were taken from multiple populations (4 populations of U. pustulata and 5 populations of D. miniatum) to minimize disturbance. U. pustulata populations were located on horizontal or gently sloping, sun-exposed rock faces. D. miniatum populations were located on vertical, wellshaded rock faces. Samples were collected and briefly cleaned of debris before being transferred to labeled paper bags before immediate transfer to a growth chamber. # Results Homologs of core circadian clock genes are present and highly conserved across lichen-forming fungal lineages. In order to investigate how widespread circadian clock-associated genes are across fungal lineages, we recovered putative homologs from the core circadian clock loop (frq, wc-1, wc-2, frh) across a wide set of lichen-forming and non-lichen-forming fungi (see . Putative homologs were included in the phylogenetic analysis after reciprocal BLAST using Neurospora crassa protein sequences. Putative homologs for lichen-forming taxa from a variety of clades were recovered, ranging from the class Lecanoromycetes to the classes Dothideomycetes and Eurotiomycetes. In order to explore potential variation between lichen-forming and non-lichen-forming fungal lineages, non-lichen-forming lineages from these classes were included when available from GenBank. Given that circadian clock homologs have been identified in lineages such as Rhizophagus irregularis in the Mucoromycota 26 and have been predicted by functional annotation in the Basidiomycota, we further included lineages in these clades in the phylogenetic analysis. Circadian homologs for wc-1 and wc-2, as well as for frq and frh, are present across most lichen-forming and select non-lichen-forming fungal clades; nonetheless, some putative homologs are missing in some lineages, including notably frq in the Trichocomaceae (e.g. Aspergillus spp. and Penicillium spp.) within the Eurotiomycetes and in several Umbilicaria spp. in the lichen-forming Lecanoromycetes [fig_ref] Figure 1: and were acclimatized for 72 h under standardized conditions of 12 [/fig_ref]. Domain architecture of putative core circadian clock homologs is broadly conserved across lichen-forming and non-lichen-forming taxa. The phylogeny inferred for circadian clock homologs of WC-1 demonstrates a broad consensus with overall fungal evolution, including the phylogenetic placement of the lichenized taxa [fig_ref] Figure 1: and were acclimatized for 72 h under standardized conditions of 12 [/fig_ref]. In order to investigate whether functional domains in the core fungal circadian clock proteins were conserved, we compared the presence of functional domains between lichen-forming and non-lichen-forming lineages in the Ascomycota as well as the Basidiomycota/Mucoromycota in the Pfam [fig_ref] Figure 1: and were acclimatized for 72 h under standardized conditions of 12 [/fig_ref] and PROSITE [fig_ref] Figure 1: and were acclimatized for 72 h under standardized conditions of 12 [/fig_ref] protein domain databases using the protein domain and visualization tool DomainViz [bib_ref] Intuitive visualization of consensus domain distributions across groups of proteins, Schläpfer [/bib_ref]. The evolution of wc-1 [fig_ref] Figure 1: and were acclimatized for 72 h under standardized conditions of 12 [/fig_ref] , wc-2, frq and frh (Figs. S1-S3) largely mirrors known class-level relationships of the fungal tree of life. Clock genes of lichen-forming taxa fall within the respective fungal classes (Lecanoromycetes, Eurotiomycetes, Dothideomycetes) with high support, rather than forming a monophyletic clade. This suggests that there are no shared signatures of lichenization in the analyzed clock genes. The results demonstrate that two key regions of the Neurospora WC-1 protein are broadly conserved between lichen-forming Ascomycota, non-lichen-forming Ascomycota, and Basidiomycota/Mucoromycota lineages. The first of these is the PAS region, originally identified in the Drosophila melanogaster period clock (PER), Ah receptor nuclear translocator (ARNT) and single-minded (SIM) proteins, and which is involved in protein dimerization, light perception, light regulation and circadian rhythm regulation. At the C-terminal end, there is broad conservation across all three investigated lineages in the GATA-type zinc finger motif [fig_ref] Figure 1: and were acclimatized for 72 h under standardized conditions of 12 [/fig_ref] , involved in protein localization to a set of consensus sequences in the regulatory regions of many light-and clock-regulated genes [bib_ref] Conservation, convergence, and divergence of lightresponsive, circadian-regulated, and tissue-specific expression patterns during..., Manfield [/bib_ref]. Light-dependent response of the core circadian clock gene frequency is conserved in two highly diverged lichen-forming fungal lineages. To investigate whether frq homologs in lichenforming fungal lineages maintain their primary circadian function as targets of the WCC, we investigated the transcript abundance of frq putative homologs in response to light in Dermatocarpon miniatum (Ascomycota, Eurotiomycetes) and Umbilicaria pustulata (Ascomycota, Lecanoromycetes), two species belonging to different Ascomycete classes [fig_ref] Figure 1: and were acclimatized for 72 h under standardized conditions of 12 [/fig_ref]. Thalli were harvested from a colocalized site in the Taunus mountains of Germany [fig_ref] Figure 2: Homologs of frq in two lichen-forming fungi are responsive to light [/fig_ref] www.nature.com/scientificreports/ following which half of the thalli were exposed to 20 min of light (30 μE) before harvesting [fig_ref] Figure 2: Homologs of frq in two lichen-forming fungi are responsive to light [/fig_ref]. All primers were previously tested for each species and no fluorescence was detected in negative controls (dH 2 0 and RT-controls). After significant gene:treatment interactions were detected in ANOVA for both species, post-hoc tests revealed significantly increased frq transcript abundance in both D. miniatum (p = 0.0012, Šidàk-adjusted contrasts) and U. pustulata (p = 0.0101, Šidàk-adjusted contrasts) after exposure to light, while transcript abundance of wc-1 was not significantly affected by the light treatment in either species [fig_ref] Figure 2: Homologs of frq in two lichen-forming fungi are responsive to light [/fig_ref]. Statistical results for each species are summarized in . # Discussion The fungal circadian system, as elucidated initially in Neurospora crassa, is composed of three primary oscillators: a FRQ/WCC-dependent oscillator (FWO) as well as FRQ-independent (WC-FLO) and oscillators that are both FRQ-and WCC-independent (FLO) [bib_ref] The diversity and evolution of circadian clock proteins in fungi, Salichos [/bib_ref].The main FRQ/WCC-dependent oscillator, of which frq, wc-1, wc-2 and frh are the core components, was initially thought to be limited to Ascomycota, but has since been identified in various lineages across the Basidiomycota and Mucoromycota. Although a frq homolog has been previously identified in the genome of Umbilicaria pustulata within the lichen-forming Lecanoromycetes [bib_ref] Circadian rhythms in fungi: Structure/function/evolution of some clock components, Brody [/bib_ref] , there have been no previous reports on the presence or absence of circadian clock genes across lichen-forming fungi more broadly. The identification of putative homologs of circadian and light-sensing genes in a wide range of lichen-forming fungal lineages, combined with the conserved light-activated function of the core circadian clock component frq in two highly diverged lichen-forming fungi, points to the conservation of the circadian clock mechanism in mycobionts. Lichen-forming fungi exist in stable symbioses with algae and cyanobacteria, both of which have relatively well-understood circadian clocks in free-living conditions [bib_ref] Resonating circadian clocks enhance fitness in cyanobacteria, Ouyang [/bib_ref] [bib_ref] The world of algae reveals a broad variety of cryptochrome properties and..., Petersen [/bib_ref] [bib_ref] Genome characteristics reveal the impact of lichenization on lichen-forming fungus Endocarpon pusillum..., Wang [/bib_ref]. The elucidation of the well-conserved fungal circadian mechanism in mycobionts thus opens the door for the investigation of how the circadian systems of myco-and photobionts interact and coordinate. Increased attention has recently been paid to holobiont chronobiology and the characteristics of circadian systems in symbiotic interactions [bib_ref] Holobiont chronobiology: mycorrhiza may be a key to linking aboveground and underground..., Lee [/bib_ref] , although the vast majority of investigations into the evolution and function of circadian clock components have been made in free-living organisms. To date, most holobiont chronobiology www.nature.com/scientificreports/ studies deal with microbiotic effects of prokaryotes and unicellular eukaryotes on host chronobiology [bib_ref] The metronome of symbiosis: Interactions between microbes and the host circadian clock, Heath-Heckman [/bib_ref] [bib_ref] Holobiont chronobiology: mycorrhiza may be a key to linking aboveground and underground..., Lee [/bib_ref]. Nonetheless, important inroads into the role of rhythmicity have been made in eukaryotic-eukaryotic systems ranging from arbuscular mycorrhizal fungal interactions and their host plants [bib_ref] Expression of putative circadian clock components in the arbuscular mycorrhizal fungus Rhizoglomus..., Lee [/bib_ref] as well as Symbiodinium algae and their coral host species [bib_ref] Circadian clocks in symbiotic corals: The duet between Symbiodinium algae and their..., Sorek [/bib_ref]. The lichen symbiosis is composed of partners with highly specialized and often wellinvestigated roles [bib_ref] Genome characteristics reveal the impact of lichenization on lichen-forming fungus Endocarpon pusillum..., Wang [/bib_ref] [bib_ref] Symbiotic interplay of fungi, algae, and bacteria within the Lung Lichen Lobaria..., Eymann [/bib_ref] , and outputs from one symbiont (such as photosynthates) may affect the production of compounds by the other symbiont [bib_ref] Photosynthates stimulate the UV-B induced fungal anthraquinone synthesis in the foliose lichen..., Solhaug [/bib_ref]. Each symbiont produces compounds in a seasonally-variable manner [bib_ref] Annual variation in photoacclimation and photoprotection of the photobiont in the foliose..., Vráblíková [/bib_ref] [bib_ref] Seasonal variation of antioxidant activity and phenolic content of Pseudevernia furfuracea, Evernia..., Aoussar [/bib_ref] , which may be regulated at least in part by the photosensory and circadian machinery of the symbionts. Lichen symbioses are also remarkably stable and geographically widespread, and the exchange of symbionts along gradients has been reported (e.g. [bib_ref] Photobiont selectivity and specificity in Caloplaca species in a fog-induced community in..., Vargas Castillo [/bib_ref] [bib_ref] Environment and host identity structure communities of green algal symbionts in lichens, Dal Grande [/bib_ref] [bib_ref] Expanding the mutualistic niche: Parallel symbiont turnover along climatic gradients, Rolshausen [/bib_ref] , providing an excellent model in which to study the effect of each partner's circadian system in the holobiont. While we identified putative homologs of all components of the core circadian clock machinery in nearly all lineages, some of the core circadian clock genes were missing in some lineages. The gene frq appears to be missing from certain isolated lichen-forming fungal lineages in the Umbilicariaceae, as well as from the Trichocomaceae (Aspergillus spp. and Penicillium spp.) within the Eurotiomycetes. The main paradigm of the evolution of frq in the Fungi has changed considerably over the last decades. Originally identified in Neurospora crassa (Ascomycota, Sordariomycetes), initial investigations of its evolution restricted its presence to within the Sordariomycetes, Leotiomycetes and Dothideomycetes within the Ascomycota 46 . More recent investigations have expanded this understanding considerably, and frq is understood to have evolved at least before the divergence of Mucoromycotina and Zoopagomycota from Dikarya [bib_ref] Circadian rhythms in fungi: Structure/function/evolution of some clock components, Brody [/bib_ref]. In our present study, the putative homologs of frq could not be found in the genomes of certain lichenforming fungal lineages (U. phaea, U. deusta, U. spodochroa, U. freyi; see for an overview of the Umbilicariaceae). Little life history or ecological rationale can at present be given for a parsimonious loss of the frq homolog in these lineages alone; interestingly, we were able to find a copy for one of these species (U. spodochroa) as well as for other lineages that do not have their genomes sequenced , using a degenerate primer design approach . It may be that frq is present in many more species but simply remains undetected in some genomes because of issues with local genome quality, genome assembly, annotation, and gene prediction. For the other circadian clock components investigated in this study, we found that frh and wc-2 were occasionally missing in single disparate lineages: wc-2 in Alternaria alternata and wc-2 and frh in Sordaria fimicola. Of the four genes investigated in this study, only the gene wc-1 was identified in every lineage without exception. The rapid induction of frq expression by light has long been a characteristic phenotype of the main FWO oscillator in N. crassa (since e.g. Crosthwaite et al. [bib_ref] Light-induced resetting of a circadian clock is mediated by a rapid increase..., Crosthwaite [/bib_ref]. This phenotype of light-induced frq expression has been observed in other Ascomycota such as the soil-living saprophyte Pyronema confluens (Pezizomycetes) [bib_ref] Analysis of circadian rhythms in the basal filamentous ascomycete Pyronema confluens, Traeger [/bib_ref] , as well as in the Mucoromycota, such as the arbuscular mycorrhizal fungus R. irregularis [bib_ref] Expression of putative circadian clock components in the arbuscular mycorrhizal fungus Rhizoglomus..., Lee [/bib_ref]. The presence of frq, wc-1, and wc-2 alone, however, is not enough to infer a functional FWO oscillator: in the plant pathogen Verticillium dahliae, the frq homolog is present, but is largely unresponsive to light [bib_ref] No evidence that homologs of key circadian clock genes direct circadian programs..., Cascant-Lopez [/bib_ref]. It is thus noteworthy that, in the present study, we observe that lichen-forming fungi from two highly diverged lineages, the Lecanoromycetes (U. pustulata) and the Eurotiomycetes (D. miniatum) both display frq light-dependent responses similar to the canonical responses of N. crassa, pointing to a functionally-conserved core circadian clock mechanism in at least two highly-diverged groups of lichen-forming fungi. Circadian rhythms are central to the functioning of symbiotic interactions, as evidenced by the squid-Vibrio 25 , coral-Symbiodinium 23,24 , and plant-mycorrhiza 26 symbioses. The lichen system is an unexplored model of symbiosis with respect to the circadian clock that has great potential in contributing to our understanding of organism-organism and organism-environment interactions. Having identified the core components of the fungal side of the lichen symbiosis and determined their functional conservation in at least two broadly-diverged lineages of lichen-forming fungi, we can now address questions like: How does the circadian system influence seasonal and diurnal dynamics in the lichen symbiosis, such as growth, nitrogen fixation, photosynthesis, or ascospore discharge? Which seasonal and diurnal rhythms are important in this mutualism in the first place? To what extent do the circadian clocks of the mycobiont and photobiont interact and interdepend? Is there circadian regulation in partner recruitment, establishment, and maintenance of the symbiotic association? What is the role of the circadian clock machinery in climatic niche specialization? An in-depth knowledge of the lichen circadian clock and its outputs and contributions to the symbiosis has the potential to enhance our understanding of symbiotic systems more broadly, from geographic distributions to interactions at the cellular and the molecular levels. . ## Data availability [fig] Figure 1: and were acclimatized for 72 h under standardized conditions of 12:12 light:dark at 16° C, Presence of core circadian clock genes in fungi and domain architecture of White Collar-1 (wc-1). (a) Phylogenetic tree based on a WC-1 sequence alignment of 31 lichen-forming fungi (green) and a selection of non-lichen-forming taxa. This is a maximum likelihood tree. Bootstrap support greater than 75% is indicated at the branches. Taxa used in the experimental part of this study are marked by asterisks (***). Presence/absence of putative circadian homologs from Neurospora crassa is shown adjacent to each species. Functional domains annotated according to Pfam (b-d) and PROSITE (e-g) databases in lichen-forming (d, g) and non-lichen-forming lineages in the Ascomycota (c, f) as well as the Basidiomycota and Mucoromycota (b, e).Scientific Reports | (2022) 12:15884 | https://doi.org/10.1038/s41598-022-19646-y [/fig] [fig] Figure 2: Homologs of frq in two lichen-forming fungi are responsive to light. (a) Thalli of the lichen-forming fungi Dermatocarpon miniatum and Umbilicaria pustulata, harvested in the Taunus mountain range of central Germany. (b) Lichen thalli were acclimatized to standard laboratory conditions via a 72 h LD 12:12 cycle at 16° C before 24 h in constant darkness, followed by a 20-min light induction at CT 0. (c) RT-qPCR analysis demonstrates broad conservation of light responsiveness in the circadian clock components of lichen-forming fungi. n = 6 for Dmin, n = 5 for Upust per treatment; mean ± SEM are shown. Asterisks represent significance extracted using Šidàk-adjusted contrasts: *P < 0.05; **P < 0.01; ***P < 0.001. Dmin: Dermatocarpon miniatum; Upust: Umbilicaria pustulata; frq: frequency; wc-1: white collar-1. Scientific Reports | (2022) 12:15884 | https://doi.org/10.1038/s41598-022-19646-y [/fig]

Measuring the impact and distress of health problems from the individual's perspective: development of the Perceived Impact of Problem Profile (PIPP) Background:The aim of this study was to develop and conduct preliminary validation of the Perceived Impact of Problem Profile (PIPP). Based on the biopsychosocial model of health and functioning, the PIPP was intended as a generic research and clinical measurement tool to assess the impact and distress of health conditions from the individuals' perspective. The ICF classification system was used to guide the structure of the PIPP with subscales included to assess impact on selfcare, mobility, participation, relationships and psychological well-being. While the ICF focuses on the classification of objective health and health related status, the PIPP broadens this focus to address the individuals' subjective experience of their health condition.Methods: An item pool of 23 items assessing both impact and distress on five key domains was generated. These were administered to 169 adults with mobility impairment. Rasch analysis using RUMM2020 was conducted to assess the psychometric properties of each set of items. Preliminary construct validation of the PIPP was performed using the EQ5D.Results: For both the Impact and Distress scales of the PIPP, the five subscales (Self-care, Mobility, Participation, Relationships, and Psychological Well-being) showed adequate psychometric properties, demonstrating fit to the Rasch model. All subscales showed adequate person separation reliability and no evidence of differential item functioning for sex, age, educational level or rural vs urban residence. Preliminary validity testing using the EQ5D items provided support for the subscales.Conclusion: This preliminary study, using a sample of adults with mobility impairment, provides support for the psychometric properties of the PIPP as a potential clinical and research measurement tool. The PIPP provides a brief, but comprehensive means to assess the key ICF components, focusing on the individuals' perspective of the impact and distress caused by their health condition. Further validation of its use across different health conditions and varying cultural settings is required. # Background There exist a number of models that provide a conceptualization of health and disability. Within the traditional medical model, disability is viewed as a characteristic of an individual that is caused by the health condition, requiring the need for medical intervention by professionals to manage the condition. The social model, in contrast, does not view disability as being a characteristic of an individual, but as a socially induced problem. The social model differentiates impairment and disability, with the term impairment referring to the physical condition and disability to the societal and environmental barriers inhibiting social participation of individuals with impairment. In 2000, the WHO introduced The International Classification of Functioning, Disability and Health (ICF), which integrates these two models and adopts a biopsychosocial approach which views biological, psychological, and social processes as integral to and interactive with physical health and illness. Within the framework of the ICF, disability and functioning are seen as the result of complex interactions between health conditions and contextual factors, both environmental and personal. Environmental factors, both physical (climate, terrain, architecture) and social (legal and social structures, cultural and social attitudes), and personal factors, such as gender, age, education, character, coping styles and past experience, may all contribute to the impact of a specific health condition on an individual. Coupled with the shift in emphasis on the social construction of disability, there has been a growing awareness of the need to measure the impact of a health condition from the individual's perspective, taking into account social context and personal factors. While an objective assessment of an individual's symptoms and his or her functional status is important, it only provides part of the picture. At a practical level, the biopsychosocial model considers that understanding the person's subjective experience is essential for accurate diagnosis, health outcomes and appropriate care. Hewlett [bib_ref] Measuring the meaning of disability in rheumatoid arthritis: the Personal Impact Health..., Hewlett [/bib_ref] has argued that difficulty in performing valued activities would determine the personal impact of a particular impairment, and consequently that it was necessary to assess the perceived importance to the individual of specific activities (eg. tasks involved in dressing and grooming, preparing and eating food, personal hygiene). These importance ratings were then used to weight the individual's disability scores on the Health Assessment Questionnaire, resulting in a personal impact score (PI HAQ) [bib_ref] Measuring the meaning of disability in rheumatoid arthritis: the Personal Impact Health..., Hewlett [/bib_ref]. The results of this study suggest that individuals with similar impairment levels (as determined by the HAQ) can have very different levels of personal impact arising from the impairment. Other researchers have taken the individual or patient focused approach to measurement a step further. 'Patient generated outcome measures' [bib_ref] A review of selected patientgenerated outcome measures and their application in clinical..., Patell [/bib_ref] or individualized questionnaires are increasingly popular in treatment outcome studies. These measures do not consist of predefined domains (eg. self-care, physical function, social interaction) but instead, individuals are asked to nominate areas or activities in their own lives that are affected by their health condition. Typically weightings are applied to scores, based on the relative importance assigned by the individual. Examples of this type of measure include the Patient Generated Index (PGI, Schedule for the Evaluation of Individual Quality of Life (SEIQOL [bib_ref] Individual quality of life in patients undergoing hip replacement, O&apos;boyle [/bib_ref] [bib_ref] A new short form individual quality of life measure (SEIQoL-DW): application in..., Hickey [/bib_ref] , and the Asthma Quality of Life Questionnaire (AQLQ [bib_ref] Validation of a standardized version of the Asthma Quality of Life Questionnaire, Juniper [/bib_ref]. While these individual or patient-generated measures provide insight into individual experience, recent reviews of such instruments have identified potential problems and disadvantages [bib_ref] A review of selected patientgenerated outcome measures and their application in clinical..., Patell [/bib_ref] [bib_ref] Why are we weighting? The role of importance ratings in quality of..., Trauer [/bib_ref]. Many patient-generated measures use weightings to assign importance to specific domains of individual's lives, but the mathematical appropriateness of weightings in quality of life measures has recently been questioned. Trauer and MacKinnon [bib_ref] Why are we weighting? The role of importance ratings in quality of..., Trauer [/bib_ref] , for example, show that multiplicative composites have a number of undesirable psychometric properties, making them unsuitable for many statistical procedures, although they are also problematic for practical reasons [bib_ref] A review of selected patientgenerated outcome measures and their application in clinical..., Patell [/bib_ref]. The measures have been criticized as time consuming and cumbersome to complete, resulting in missing data, and they require complex judgement tasks beyond the comprehension of some respondents with limited education or cognitive impairment. In addition, lack of standardization means that comparisons across individuals with different health conditions or across time points for the same individual are not possible. Patel et al. [bib_ref] A review of selected patientgenerated outcome measures and their application in clinical..., Patell [/bib_ref] concludes that they are not suitable for use in clinical trials or treatment evaluation, but may be better suited in the consultative process to design a therapy plan. ## Current study In this paper, we report on the development and preliminary validation of an instrument designed to overcome some of the weaknesses of patient-generated measures, while still focusing on the impact of health condition from the individual's perspective. The instrument was designed to be generic rather than disease specific, making it suitable for use across different groups. Unlike existing measures of functioning, the instrument was not designed to assess an individual's ability to perform certain tasks or activities, but rather to better understand the impact and distress caused by the health condition. This was intended to supplement more objective measures (eg. SF-36and the Health Assessment Questionnaire [bib_ref] Measurement of patient outcome in arthritis, Fries [/bib_ref] , providing additional information incorporating contextual factors (environment and personal) identified in the ICF. This information can play an important role in optimising the provision of health care services and setting of priorities. For example, two patients with similar health problems (eg. restricted movement in the knee joint) may experience very different levels of impact and distress. Respondent 1, a professional footballer, may experience highly elevated levels of impact and distress concerning his condition, requiring immediate treatment. In comparison, Respondent 2, a 75 year old with a sedentary life style, may report relatively little impact or distress. This understanding of the meaning of the health condition for the individual can be used in clinical settings to guide the development of treatment programs and to assess outcomes and effectiveness. Our aim in this study was to develop a relatively short, self-report instrument to assess both the impact and the distress of health problems from the individual's perspective. A set of standardized domains was specified to allow comparison of scores across patient groups and in individuals over multiple time points. The selection of domains for inclusion was guided in part by the ICF, a review of existing measures, and a series of qualitative interviews. Items were selected to assess the impact and distress of the health condition on the Activities and Participation domains of the ICF (Chapters 4 to 9). An additional component was included to assess the impact on the individual's psychological well-being (including independence and autonomy), an area not yet well addressed in the ICF. ## Development of the perceived impact of problem profile (pipp) A general test plan was developed representing five separate subscales (Self-care, Mobility, Participation, Relationships, Psychological Well-being) (see [fig_ref] Table 1: PIPP items and corresponding ICF codes [/fig_ref] for items and corresponding ICF code). Prior to the generation of specific items, qualitative interviews were conducted to identify aspects of individuals' lives that they felt were affected by their disability and caused them distress or reduced their perceived quality of life, to ensure that all relevant aspects were identified for inclusion in the scale. A total item pool of 23 items was generated representing the key domains identified (see [fig_ref] Table 1: PIPP items and corresponding ICF codes [/fig_ref]. For each item, respondents were asked to rate on a 6-point scale (a) 'how much impact has your current health problems had on [item of function or activity]'; and (b) 'How much distress has been caused by the impact of your health problem on [same item of function or activity]'. The 6-point scale was anchored on either end by 'no impact' and 'extreme impact' for the Impact scale and by 'no distress' and 'extreme distress' for the Distress scale. For each item, an additional response option was provided for the individual to indicate that the activity was not applicable. The wording of the items was carefully chosen to ensure that the activities were suitable for both males and females, across different age groups, and for use in different cultural contexts. This study was designed as part of a larger cross-cultural project (RESILIENCE project), with the development of items being conducted in collaboration with researchers from Malaysia and Thailand. The initial version of the PIPP was pilot tested in a series of interviews with individuals with mobility impairment in Australia, Malaysia and Thailand. ## Validation sample with locomotive disorders Musculoskeletal conditions are the most common cause of physical disability and severe, chronic pain, affecting millions of people globally. These include a wide spectrum of conditions, including those of acute onset (e.g. trauma, injury), and short or long-term disorders (e.g. arthritis, multiple sclerosis). Due to its frequency and chronicity, and the degree of disability that can result, it has a pervasive impact on society. The frequency of musculoskeletal conditions increases with age, and with longer life-spans and hence, ageing populations, there will be an increased number of people with chronic disability disorders with increasing requirements to access healthcare services. A recommended ICF core set for people with musculoskeletal conditions in acute care included items from the Activities and Participation component such as mobility, self-care, interpersonal interactions, relationships, handling stress and psychological demands [bib_ref] ICF Core Set for patients with musculoskeletal conditions in the acute hospital, Stoll [/bib_ref]. In fact, mobility (walking) was found to have reached a consensus of at least 80% as an important and relevant domain among a range of health conditions, including low back pain, osteoporosis, rheumatoid arthritis, osteoarthritis, chronic ischaemic heart disease and diabetes mellitus [bib_ref] Identification of relevant ICF categories in patients with chronic health conditions: a..., Weigl [/bib_ref]. The preliminary validation of the PIPP was conducted using a sample of people with mobility impairment. The impact of mobility impairment on life quality is multidimensional, affecting individuals by limitations in activity and restrictions in participation. Subsequent consequences can include reduced employment potential and income, lifestyle limitations, decreased ability to carry out self-care activities, increased dependency on others, and poorer quality of life. Hence, mobility impairment can have a profound impact on the psychosocial status of the individual. # Methods ## Participants Participants were recruited through co-operation with the Central Highlands General Practice Division. Employing a two staged modified cluster sampling [bib_ref] Cluster sampling to assess immunization coverage -A review of experience with a..., Henderson [/bib_ref] [bib_ref] A computer simulation of household sampling schemes for health surveys in developing..., Bennett [/bib_ref] , 30 gen-eral practitioners in the Central Highlands region were selected. Each practitioner was asked to select seven adult patients from his or her practice who, for any reason, experienced impaired mobility or problems with walking. Rather than diagnostic categories, a liberal criterion was used to identify potential participants, and reasons for mobility impairment included limb amputation (e.g. from accident, cancer or vascular disease), spinal cord injury, stroke, degenerative conditions (e.g. arthritis), and diseases affecting the central nervous system (e.g. multiple sclerosis). While the aim was to recruit 210 across the 30 practices, initially 178 participants were recruited, who gave verbal consent to the general practitioner or practice nurse and were then contacted by a member of the research team. Twenty-one of these participants declined to proceed for various reasons including personal health (e.g. too ill to partake in the interview), and health of family member (e.g. nursing dying husband), while others were unable to keep appointments despite visits by the research team on more than two occasions. Advertisements were placed in a few local newspapers to increase the number of participants and an additional 12 participants were recruited using this approach. In total, 169 participants were recruited and interviewed in person for this study. Ethics approval for the study was granted by the Human Research Ethics committee of The University of Melbourne. # Materials Additional questions and scales were included in the questionnaire battery to allow an exploration of the validity of the PIPP. These included demographic questions, some details concerning their health condition, and the EQ5D. The EQ5D, developed by the EuroQol Groupis a brief standardized, non-disease-specific instrument to measure physical, emotional and social functioning and well-being, and has been used extensively worldwide. It assesses five dimensions -mobility, self-care, usual activities, pain/discomfort, and anxiety/depression. Within each domain, there are three possible responses: 'no problems,' 'some problems,' and 'unable to perform.' These five dimensions of health are complemented by a 20-cm vertical visual analogue acale (EQ5D VAS) for the respondent to rate his or her health today from the 'best imaginable health state' set at 100 and 'worst imaginable health state' set at 0. The EQ5D was used with the permission of the EuroQoL Group. ## Statistical analyses The PIPP Impact and PIPP Distress responses were subjected to Rasch analysis using the RUMM2020 softwareto assess the psychometric properties of each set of items. For each of the individual subscales, a series of analyses was undertaken to assess overall model fit, threshold ordering, item fit, person fit, and differential item functioning. Rasch calibrated subscale scores were then exported into SPSS Version 12 for further statistical analyses to assess the validity of the subscales. The construct validity of the subscales was assessed in relation to the corresponding component of the EQ5D which was included in the questionnaire booklet. Further details of the Rasch procedures undertaken are provided in the section to follow; a detailed description and illustration of the Rasch analysis procedures is provided in Pallant and Tennant [bib_ref] An introduction to the Rasch measurement model: An example using the Hospital..., Pallant [/bib_ref]. The RUMM2020 softwarewas used to assess how well the observed data fit the expectations of the Rasch measurement model. The Rasch model provides a template that operationalizes the formal axioms that underpin measurement [bib_ref] The Rasch model, additive conjoint measurement, and new models of probabilistic measurement..., Karabatos [/bib_ref]. Initially the appropriateness of the response scale was assessed by inspection of the threshold values for each item. The term threshold refers to the point between two response categories where either response is equally probable. For a good fitting model, we would expect that, for each of the items, respondents with high levels of the attribute being measured would endorse high scoring responses, while individuals with low levels of the attribute would consistently endorse low scoring responses. This would be indicated by an ordered set of response thresholds for each of the items. One of the most common sources of item misfit concerns respondents' inconsistent use of these response options, resulting in what is referred to as disordered thresholds. This suggests that respondents have difficulty consistently discriminating between response options. Collapsing of categories was used to correct disordered thresholds to improve overall fit to the model. Three overall fit statistics were considered to assess model fit. Two are item-person interaction statistics transformed to approximate a z-score, representing a standardized normal distribution. If the items and persons fit the model, we would expect to see a mean of approximately zero and a standard deviation of 1. The third fit statistic was an item-trait interaction statistic reported as a chi-square, reflecting the property of invariance across the trait. A significant chi-square indicates that the hierarchical ordering of the items varies across the trait, so compromising the required property of invariance. Individual person-fit and item-fit statistics were also assessed, both as residuals (a summation of individual person and item deviations) and as a chi-square statistic. Residuals ± 2.5 are considered to indicate adequate fit to the model. The summed chi-square within each group provides the overall chi-square for the item, and the overall chi-square for items is summed to give the item traitinteraction statistic. Bonferroni corrections are applied to adjust the chi-square p value to take account of multiple testing [bib_ref] Multiple significance tests: The Bonferroni method, Bland [/bib_ref]. An estimate of the internal consistency reliability is provided as a Person Separation Index (PSI), where the estimates on the logit scale for each person are used to calculate reliability. PSI values can be interpreted in the same way as Cronbach alpha coefficients, with values above .7 considered adequate. Assessment of differential item functioning (DIF) was used to identify possible item bias. This can occur when different groups within the sample (e.g. males and females), despite equal levels of the underlying character-istic being measured, respond in a different manner to an individual item. Two types of DIF were assessed statistically and graphically using the RUMM2020 software. Uniform DIF exists when one group shows a consistent systematic difference in its responses to an item, across the whole range of the attribute being measured. When there is non-uniformity in the group differences (e.g. it varies across levels of the attribute), then this is referred to as non-uniform DIF. Analysis of variance was conducted for each item, comparing scores across each level of the 'person factor' and across different levels of trait (referred to as class intervals). Uniform DIF is indicated by a significant main effect for the person factor (gender), while the presence of non-uniform DIF is indicated by a significant interaction effect (person factor × class interval). # Results Separate analyses are reported for the PIPP Impact and PIPP Distress items. For each set of items, Rasch analysis was conducted for the individual subscales of Self-care, Mobility, Participation, Relationships and Psychological Well-being. ## Pipp impact subscales Prior to assessing the model fit of the subscales, the threshold ordering of the items was inspected. All items showed some degree of disordering, except item 9 (sit or stand). To overcome this problem, all items were recoded from the original 6-point scale (scored: 012345) by collapsing scores to form a simpler 3-point response scale (011112). This resulted in ordered thresholds for all items except item 14 (authority), which showed only minor disordering. ## Pipp impact: self-care The four self-care items included in the Impact scale were subjected to Rasch analysis to assess their ability to form a psychometrically sound subscale. The overall model fit statistics were non-significant, indicating no serious misfit to the model (item-trait interaction chi-square = 10.61, df = 8, p = .23). The PSI was .79, suggesting adequate person separation reliability, given the small number of items involved. The mean fit residual value for items was -.13 with a standard deviation (SD) of .20, while the mean fit residual for persons was -.39 with a SD of .76. No misfitting items were detected, with all individual item fit statistics being non significant (see [fig_ref] Table 2: Individual item fit statistics for PIPP [/fig_ref]. Items were found to be free of differential item functioning for sex, age, education level and rural/urban residence. ## Pipp impact: mobility Rasch analysis of the five mobility items revealed no disordered thresholds (after rescoring 011112), good overall model fit (item-trait interaction chi-square = 11.45, df = 10, p = .32) and adequate person separation reliability (PSI = .75) (see [fig_ref] Table 2: Individual item fit statistics for PIPP [/fig_ref]. The mean fit residual value for items was .16 (SD = .95), while the mean fit residual for persons was -.36 (SD = 1.02). There were no misfitting items and no evidence of differential item functioning. ## Pipp impact: participation Initial inspection of the overall model fit statistics for the five participation items indicated some misfit to the model (item-trait interaction chi-square = 26.7, df = 10, p = .003) (see [fig_ref] Table 2: Individual item fit statistics for PIPP [/fig_ref]. All thresholds were ordered (after global recoding to 011112), however individual item fit statistics revealed one item (item 8: assist other family members) with a positive fit residual exceeding 2.5 (2.775). After removal of this item from the scale the overall fit statistics improved (item-interaction chi-square = 7.28, df = 8.0, p = .51). The mean fit residual value for items was .34 (SD = .68), while the mean fit residual for persons was -.29 (SD = 1.05). The final PSI was .78, indicating adequate person separation reliability. There was no evidence of differential item functioning for sex, age, education level or rural/urban residence. ## Pipp impact: relationships Analysis of the four relationship items revealed a non-significant item-trait chi-square statistic (chi-square = 11.90, df = 8, p = .16), indicating adequate fit to the model, with no misfitting items (see [fig_ref] Table 2: Individual item fit statistics for PIPP [/fig_ref]. The mean fit residual value for items was .61(SD = .32), while the mean fit residual for persons was -.23 (SD = 1.13). Items were free of differential item functioning. The Person Separation Index for this subscale was low (.69), due primarily to the fact that there was considerable missing data for the item concerning close relationships. Inspection of the targeting map revealed a floor effect with a clustering of respondents reporting relatively little impact of their health condition on relationships (see . ## Pipp impact: psychological well-being Rasch analysis of the five items relating to psychological well-being showed good fit to the model (item-trait chisquare = 8.43, df = 10, p = .59) with an adequate PSI value of .73 (see [fig_ref] Table 2: Individual item fit statistics for PIPP [/fig_ref]. The mean fit residual value for items was -.60 (SD = .71), while the mean fit residual for persons was -.74 (SD = 1.4). Inspection of the individual item fit statistics identified no misfitting items or differential item functioning. ## Pipp distress subscales Consistent with the procedures adopted in the previous section, the items from each of the individual subscales were subjected to Rasch analysis. Disordered thresholds were corrected by recoding all items as 011112. ## Pipp distress: self-care The overall model fit statistics for the four Distress Selfcare items were non-significant, indicating no serious misfit to the model (item-trait interaction chi-square = 9.42, df = 8, p = .31) (see [fig_ref] Table 3: Individual item fit statistics for PIPP [/fig_ref]. The mean fit residual value for items was .12 (SD = .53), while the mean fit residual for persons was -.29 (SD = .80). The PSI was .79, suggesting adequate person separation reliability, given the small number of items involved. No misfitting items were detected, with all individual item fit statistics being non significant (see [fig_ref] Table 3: Individual item fit statistics for PIPP [/fig_ref]. Items were found to be free of differential item functioning for sex, age, education level and rural/urban residence. ## Pipp distress: mobility The five Distress Mobility items showed adequate overall model fit (item-trait interaction chi-square = 19.06, df = 10, p = .04) and good person separation reliability (PSI = .83). The mean fit residual value for items was .13 (SD = .76), while the mean fit residual for persons was -.46 (SD = 1.32). Inspection of the individual item fit statistics (see [fig_ref] Table 3: Individual item fit statistics for PIPP [/fig_ref] however revealed a misfitting item (item 12: abil-ity to move around own house). While removal of this item from the subscale resulted in an improvement in the overall model fit (item-trait interaction chi square = 12.06, df = 8, p = .15), it resulted in a substantial reduction in the PSI, from .83 to .78. It was therefore decided to retain this item in the subscale. There was no evidence of differential item functioning for any of the mobility items. ## Pipp distress: participation The five Distress Participation items showed good overall fit to the Rasch (item-trait interaction chi-square = 14.23, df = 10, p = .16) with a PSI of .79 (see [fig_ref] Table 3: Individual item fit statistics for PIPP [/fig_ref]. All thresholds were ordered, there were no misfitting items, and no evidence of differential item functioning. The mean fit residual value for items was .20 (SD = 1.14), while the mean fit residual for persons was -.41 (SD = 1.35). ## Pipp distress: relationships Analysis of the four Distress Relationship items revealed a non-significant item-trait chi-square statistic (chi-square = 8.82, df = 8, p = .36) indicating adequate fit to the model, with no misfitting items (see [fig_ref] Table 3: Individual item fit statistics for PIPP [/fig_ref]. The mean fit resid- ual value for items was .54 (SD = .41), while the mean fit residual for persons was -.25 (SD = 1.19). The PSI for this subscale was adequate (.73), however there was considerable missing data for the item concerning close relationships. Items were found to be free of differential item functioning. ## Pipp distress: psychological well-being Rasch analysis of the five items relating to psychological well-being showed good fit to the model (item-trait chisquare = 5.66, df = 10, p = .84) with a good PSI value of .83 (see [fig_ref] Table 3: Individual item fit statistics for PIPP [/fig_ref]. The mean fit residual value for items was -.47 (SD = .49), while the mean fit residual for persons was -.76 (SD = 1.55). Inspection of the individual item fit statistics identified no misfitting items or differential item functioning. ## Validation of pipp subscales To further assess the characteristics of the PIPP subscales, the person estimates generated in RUMM2020 for each of the subscales were exported to an SPSS data file. These were then subjected to further statistical analyses to assess the relationship among the PIPP subscales, between the impact and distress subscales, and between the subscales and the various components of the EQ5D, which were included in the questionnaire booklet. The descriptive statistics for the Impact and Distress subscales are shown in [fig_ref] Table 4: Descriptive statistics for Rasch calibrated scores of PIPP Impact and Distress subscales [/fig_ref]. Non-parametric correlation coefficients (Spear-man rho) were used given the skewed distribution of scores on a number of the subscales. [fig_ref] Table 5: Spearman correlation coefficients among PIPP [/fig_ref] shows the Spearman correlation coefficients (rho) among the PIPP Impact subscales. The strongest correlation was between the impact on Mobility and Psychological Well-being (rho = .61) with the lowest occurring between Self-care and Participation (rho = .206). The correlations among the distress subscales are shown in [fig_ref] Table 6: Spearman correlation coefficients among PIPP [/fig_ref]. Overall the correlations among the distress subscales were higher than that observed for the Impact items. The highest inter-correlation was between Mobility and Psychological Well-being (rho = .78), with the lowest between Self-care and Relationship (rho = .365). ## Intercorrelations among pipp impact and distress subscales Correlation coefficients were also calculated between the corresponding PIPP Impact and PIPP Distress subscales to explore the relationship between respondents' perceptions of the impact of their disability, and the level of distress that this causes (see [fig_ref] Table 7: Spearman correlations between PIPP [/fig_ref]. All correlations were above .753 (Mobility), with the highest being recorded for the Relationship subscales (rho = .928). ## Relationship with eq5d The validity of the PIPP impact and distress subscales was assessed by investigating the relationship with appropri-Targeting map for PIPP Impact: Relationship subscale Targeting map for PIPP Impact: Relationship subscale. ate corresponding EQ5D items which were included in the questionnaire booklet administered to participants. ## Pipp psychological well-being To assess the construct validity of the PIPP Psychological Well-being, subscale scores were compared to those obtained for the EQ5D Anxiety/Depression item. Due to the small number in the extremely anxious/depressed EQ5D response category, respondents were collapsed into two groups: (1) not anxious/depressed (N = 113); and (2) moderately or extremely anxious/depressed (N = 56). Mann-Whitney tests were conducted to compare the scores for the two groups on the PIPP Impact and PIPP Distress Psychological Well-being subscales. There was a highly statistically significant difference between respondents in the two EQ5D groups for both the PIPP Psychological Well-being Impact subscale (z = -3.967, p < .001) and the PIPP Psychological Well-being Distress subscale (z = - ## Pipp self-care The PIPP Self-care subscales were compared with the EQ5D item concerning self-care. Due to the small numbers of respondents in the 'unable' response category of the EQ5D Self-care item respondents were collapsed into two categories: (1) no problems (N = 96), and (2) some problems or unable to care for self (N = 73). Mann-Whitney tests revealed significant differences between the two groups on the PIPP Self-care Impact (z = -6.789, p < .001) and PIPP Self-care Distress subscales (z = -5.106, p < .001). The mean rank scores on each PIPP subscale was substantially higher for the respondents classified as having self-care problems on the EQ5D ## Eq5d vas In addition to the individual domain items, the EQ5D VAS asks respondents to rate their health in general today on a scale from 0 to 100. The correlations between this rating and each of the PIPP Impact and Distress subscales Rasch calibrated scores are shown in [fig_ref] Table 8: Spearman correlations between PIPP scales and EQ5D 'Health in general today' item [/fig_ref]. Correlations ranged from -.20 (Impact Relationship and Distress Relationships) to -.388 (Impact Psychological Well-being). # Discussion The aim of this study was to develop, and conduct preliminary validation, of a multidimensional generic measure of the impact and distress of health conditions from the individual's perspective. For both the Impact and Distress domains of the PIPP, the five subscales (Self-care, Mobility, Participation, Relationships, Psychological Wellbeing) showed adequate psychometric properties, dem- All correlations significant at p < .01. The results of this study indicated a floor effect in regard to the Relationship subscale, with a clustering of respondents recording low scores suggesting that their health condition had little impact on their relationships. However, the correlation between impact and distress subscales for relationships was the highest among all domains (rho = 0.928). This suggests that while relationships are comparatively more resistant to the impact of mobility impairment then other domains, when a health condition does actually impact upon relationships, it results in considerable distress. The importance of relationships in terms of rehabilitation and coping with a health condition has been well documented in the literature. One of the major strengths of the PIPP is that it measures both impact and distress in regard to a variety of domains, providing valuable information on the individual's experience of their health condition. Overall, the correlations for the subscales were stronger between the distress items, All correlations significant at p < .01. as compared to the impact items. Among the impact subscales, the strongest association was between mobility and psychological well-being, and mobility and self-care. Among the distress subscales, the strongest correlations were between mobility, participation, and psychological well-being. The relationship between mobility and the other subscales for both impact and distress indicate the pervasive impact of mobility impairment, and supports its inclusion into ICF core sets for a range of different health conditions [bib_ref] Identification of relevant ICF categories in patients with chronic health conditions: a..., Weigl [/bib_ref]. Correlations between the impact and distress scores for each of the subscales ranged from .75 to .93, indicating substantial overlap between these two aspects. Exploration of the dimensionality of the set of PIPP subscales is needed, including an assessment of the appropriateness of combining subscales to provide a simpler global score for use in research contexts. Further research is also needed to assess the potential use of the detailed profile of PIPP impact and distress scales in clinical contexts. Preliminary validity testing using the EQ5D items and VAS health rating showed support for both the PIPP Impact and Distress subscales. While the EQ5D measures how much difficulty a person has in relation to various domains, the PIPP measures the subsequent impact and distress related to similar domains. In this study, those who indicated higher levels of anxiety/depression on the EQ5D were more likely to report higher impact and distress in regard to their psychological well-being. Similarly, those who had more difficulty in regard to self-care, mobility, and participation as measured by EQ5D, were more likely to also report higher impact and distress in the equivalent PIPP subscales. The ICF was used in this study to guide the conceptualization and structure of the PIPP with the inclusion of subscales representing many of the ICF elements that have been proposed as core domains for a wide spectrum of serious health conditions. While the ICF focuses on the classification of objective health and health related status, the PIPP broadens this focus to address the subjective experience of health from the individuals' perspective. In addition to the ICF elements of self-care, mobility, participation and relationships, the PIPP also incorporated items relating to psychological well-being. While these aspects are not included in the current ICF framework, these areas have been marked by the WHO for further development with the intention of 'establishing links with quality of life concepts and the measurement of subjective well-being'. Consistent with the biopsychosocial model, the PIPP provides an integrated tool with items tapping the physical, social and psychological impact and distress of a health condition from the individual's perspective. It is designed to supplement existing objective measures of functioning that focus on the individual's ability to perform certain tasks or activities. As a generic measure, rather than disease specific, it is potentially useful (subject to further psychometric assessment) for use across a range of health conditions. In this initial validation study the focus has been on the impact and distress experienced by individuals with mobility impairments. Further research is necessary to test the applicability of the PIPP across different health conditions and in different cultural contexts. While this study was conducted on a relatively small Australian sample, it forms part of a larger cross-cultural study that explores the impact and distress related to mobility in different countries, including Thailand, Malaysia, Myanmar, and Laos. Therefore, further validation of PIPP will be conducted within and across the different countries. Further investigation is also warranted concerning the potential use of the PIPP in clinical and rehabilitation settings. It may provide clinicians with a measurement tool supplying valuable information concerning the experience of the individual client, guiding the planning of therapeutic interventions and assessment of treatment outcome. Copies of the PIPP may be obtained free of charge for noncommercial use from the corresponding author. [table] Table 1: PIPP items and corresponding ICF codes [/table] [table] Table 2: Individual item fit statistics for PIPP: Impact scale items [/table] [table] Table 4: Descriptive statistics for Rasch calibrated scores of PIPP Impact and Distress subscales [/table] [table] Table 3: Individual item fit statistics for PIPP: Distress scale items [/table] [table] Table 6: Spearman correlation coefficients among PIPP: Distress subscales [/table] [table] Table 5: Spearman correlation coefficients among PIPP: Impact subscalesInitially it was necessary to collapse the original 6-point response scale to a simpler 3-point response scale to resolve disordered thresholds. The presence of disordered thresholds suggests that the respondents were unable to reliably differentiate the original six response points. Before recommending a change to the response format of the PIPP, this finding requires further investigation in other studies across different health conditions, and different cultural contexts. The simpler 3-point response scale (which is commonly used in other health scales, such as the EQ5D[15]) may have the advantage of reducing the cognitive complexity of the task of completing the PIPP ratings. In this case the use of three response points could be labelled: no impact/distress, moderate impact/dis-In order to achieve satisfactory fit to the Rasch model it was necessary to remove one item of the PIPP Impact: Participation scale (item 8: assist other family members). Removal of this item improved the fit statistics of the subscale, indicating that it was not appropriate to retain this item in the subscale in the current study. It is interesting to note that there was no evidence of misfit in the corresponding item in the PIPP Distress: Participation scale.Given the relatively small sample involved in this study the generalisability of this finding should be further investigated in larger studies, involving different health conditions. [/table] [table] Table 8: Spearman correlations between PIPP scales and EQ5D 'Health in general today' item [/table] [table] Table 7: Spearman correlations between PIPP: Impact and PIPP: Distress items [/table]

Pex18p and Pex21p, a Novel Pair of Related Peroxins Essential for Peroxisomal Targeting by the PTS2 Pathway We have identified ScPex18p and ScPex21p, two novel S . cerevisiae peroxins required for protein targeting via the PTS2 branch of peroxisomal biogenesis. Targeting by this pathway is known to involve the interaction of oligopeptide PTS2 signals with Pex7p, the PTS2 receptor. Pex7p function is conserved between yeasts and humans, with defects in the human protein causing rhizomelic chondrodysplasia punctata (RCDP), a severe, lethal peroxisome biogenesis disorder characterized by aberrant targeting of several PTS2 peroxisomal proteins, but uncertainty remains about the subcellular localization of this receptor. Previously, we have reported that ScPex7p resides predominantly in the peroxisomal matrix, suggesting that it may function as a highly unusual intraorganellar import recep-tor, and the data presented in this paper identify Pex18p and Pex21p as key components in the targeting of Pex7p to peroxisomes. They each interact specifically with Pex7p both in two-hybrid analyses and in vitro. In cells lacking both Pex18p and Pex21p, Pex7p remains cytosolic and PTS2 targeting is completely abolished. Pex18p and Pex21p are weakly homologous to each other and display partial functional redundancy, indicating that they constitute a two-member peroxin family specifically required for Pex7p and PTS2 targeting. O ne of the central tenets of peroxisomal biogenesis is that soluble peroxisomal proteins are posttranslationally imported from the cytosol into preexisting organelles [bib_ref] Biogenesis of peroxisomes, Lazarow [/bib_ref]. The identification, both in yeasts and mammals, of multiple distinct genetic complementation groups defective in peroxisomal biogenesis ( pex 1 mutants) has intimated that this is a complex process involving numerous cellular proteins (reviewed in [bib_ref] PEX genes on the rise, Subramani [/bib_ref] [bib_ref] Peroxisomes: organelles at the crossroads, Erdmann [/bib_ref] [bib_ref] Components involved in peroxisome import, biogenesis, proliferation, turnover, and movement, Subramani [/bib_ref]. Careful analysis of these mutants over recent years has revealed that peroxisomal protein import follows a branched pathway [bib_ref] Peroxisomal biogenesis: multiple pathways of protein import, Purdue [/bib_ref] , with each branch representing an import receptor specific for one of several types of peroxisome targeting sequences (PTS), followed by a shared membrane translocation process. So far, two classes of PTS have been well characterized; the COOHterminal PTS1, which is generally a tripeptide conforming to the consensus S/A/C-K/R/H-L [bib_ref] A conserved tripeptide sorts proteins to peroxisomes, Gould [/bib_ref] although other variants exist, such as the tetrapeptide Ϫ KANL PTS1 of human catalase; [bib_ref] Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal..., Purdue [/bib_ref] , and the NH 2 -terminal PTS2 oligopeptide found on several peroxisomal proteins including thiolase [bib_ref] Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable..., Osumi [/bib_ref] [bib_ref] A novel, cleavable peroxisomal targeting signal at the amino-terminus of the rat..., Swinkels [/bib_ref] [bib_ref] The peroxisomal targeting signal of 3-oxoacyl-CoA thiolase from Saccharomyces cerevisiae, Erdmann [/bib_ref] [bib_ref] Mutagenesis of the amino targeting signal of Saccharomyces cerevisiae 3-ketoacyl-CoA thiolase reveals..., Glover [/bib_ref]. Analysis of pex mutants that map to the specific branches of peroxisomal biogenesis, and thus fail to package only a subset of peroxisomal proteins, has proven particularly valuable, leading to the identification and characterization, in both yeasts and humans, of Pex5p [bib_ref] PAS10 is a tetratricopeptide-repeat protein that is essential for the import of..., Van Der Leij [/bib_ref] [bib_ref] Mutations in the PTS1 receptor gene, PXR1, define complementation group 2 of..., Dodt [/bib_ref] [bib_ref] Pay32p of the yeast Yarrowia lipolytica is an intraperoxisomal component of the..., Szilard [/bib_ref] [bib_ref] The Pichia pastoris peroxisomal protein PAS8p is the receptor for the C-terminal..., Terlecky [/bib_ref] [bib_ref] Pex20p of the yeast Yarrowia lipolytica is required for the oligomerization of..., Titorenko [/bib_ref] and Pex7p [bib_ref] PAS7 encodes a novel yeast member of the WD-40 protein family essential..., Marzioch [/bib_ref] [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] [bib_ref] Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic..., Braverman [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a..., Motley [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue..., Purdue [/bib_ref] [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref] , the import receptors for PTS1 and PTS2 type signals, respectively. Pex7p, which has now been cloned from three yeasts and also from humans and mice, is of significant interest for two main reasons. First, human PEX7 has been recently shown to be the defective gene in rhizomelic chondrodysplasia punctata (RCDP), classifying this severe and relatively common disorder of peroxisomal biogenesis as a disease of protein targeting, and emphasizing the critical role of PTS2 targeting in peroxisomal biogenesis [bib_ref] Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic..., Braverman [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a..., Motley [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue..., Purdue [/bib_ref]. Second, Pex7p is an unusual receptor in that it is soluble; this has led to considerable debate and interest regarding its subcellular distribution and function. Previously, we have reported that, when expressed in S . cerevisiae at a level equivalent to wild-type PEX7 , a COOH-terminally tagged version of ScPex7p is functional (in that it rescues growth of a ⌬ pex7 strain on oleic acid) and localized principally, if not exclusively, within the peroxisomal matrix [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref]. This surprising finding suggests that Pex7p functions as a highly unusual, intraorganellar import receptor. Pex7p import into peroxisomes is saturable; overexpressed tagged Pex7p accumulates in the cytosol as well as peroxisomes. Other researchers have reported that Pex7p is both peroxisomal and cytosolic [bib_ref] PAS7 encodes a novel yeast member of the WD-40 protein family essential..., Marzioch [/bib_ref] [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref] , and this has led to an alternative hypothesis for Pex7p function, which suggests that this is a mobile receptor that cycles between cytosol (where it can collect PTS2 cargo) and the peroxisomal surface, or matrix, where cargo is released, and Pex7p returns to the cytosol to begin another round of import. To better understand the PTS2 branch of peroxisomal biogenesis, and in doing so help unravel the mysteries of Pex7p localization and function, we have initiated a search for other proteins involved in this pathway. In this paper, we report the identification and initial characterization of Pex18p and Pex21p, a structurally and functionally related pair of peroxins that interact with Pex7p, and are specifically required for both peroxisomal biogenesis of Pex7p and PTS2 targeting. # Materials and methods ## Yeast strains , media , and growth conditions Yeast strains are shown in . For transformation, genomic DNA extraction, and preparation of yeast proteins for affinity chromatography (see below), yeast were grown in synthetic complete (SCD) medium (0.67% yeast nitrogen base, 2% dextrose supplemented with amino acids, uracil and adenine) to a density of 2 ϫ 10 7 cells/ml. For two-hybrid library screening, bait plasmid was introduced first, and these cells were grown in SCD medium lacking tryptophan (to maintain the plasmid) to a density of 2 ϫ 10 7 cells/ml, then diluted fourfold into YPD (1% yeast extract, 2% peptone, 2% dextrose) and regrown to 2 ϫ 10 7 cells/ml before transformation with library DNA. For induction of peroxisomes, cells were grown as previously described [bib_ref] Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal..., Purdue [/bib_ref] , except that preculturing was routinely performed in SCD lacking appropriate amino acids and/ or uracil, to maintain plasmids introduced into the yeast cells, before growth in YPGO medium (1% yeast extract, 2% peptone, 3% glycerol, 0.1% oleic acid, 0.25% Tween 40) or SCEO (0.67% yeast nitrogen base, 2% ethanol, 0.1% oleic acid, 0.25% Tween 40 supplemented with amino acids, uracil and adenine) for 18 h. Growth with oleic acid as sole carbon source was in SCO (0.67% yeast nitrogen base, 0.1% oleic acid, 0.25% Tween 40 supplemented with amino acids, uracil and adenine) liquid and solid media. ## Two-hybrid library screening Cells were grown as described above. To maximize transformation efficiencies, cells pretransformed with bait were processed using a carefully optimized transformation procedure [bib_ref] Transforming yeast with DNA, Gietz [/bib_ref]. Transformation efficiency was assessed by plating small aliquots onto SCD plates lacking tryptophan and leucine (to select for bait and library plasmids, respectively), and the remainder was screened on SCD plates lacking tryptophan, leucine, and adenine. Growth on these plates indicated the presence of bait and library plasmids and production of a functional Gal4p hybrid, since the ADE2 gene in PJ69-4A (which lacks Gal4p) is under the control of the Gal4p dependent GAL2 promoter (see ; [bib_ref] Genomic libraries and a host strain designed for highly efficient two-hybrid selection..., James [/bib_ref]. Positives were rescreened for activation of expression of HIS3 (by plating on media lacking tryptophan, leucine and histidine) and ␤ -galactosidase (by enzymatic assayson glass-bead homogenates), which are under the regulation of the alternative Gal4p-dependent promoters GAL1 and GAL7 in PJ69-4A, and therefore serve as additional markers of interaction [bib_ref] Genomic libraries and a host strain designed for highly efficient two-hybrid selection..., James [/bib_ref]. To isolate library plasmids from positive clones, cells were grown in SCD liquid media lacking leucine (to induce loss of bait, but not library plasmid), and plasmid DNA was prepared and transformed at low dilution into competent E . coli XL1-Blue cells for amplification and purification. Dideoxy sequencing was performed using oligonucleotides GAD424A (5 Ј -TCGATGAT-GAAGATACCCC-3 Ј ) and GAD424B (5 Ј -GCACGATGCACAGTTG-AAGTG-3 Ј ), which flank the cloning site of the library vectors. Clone identification was performed by searching the entire yeast genome database with these sequences using BLAST. ## Plasmids , cloning , and gene disruption After determination that their gene products were peroxins , yeast open reading frames YHR160C and YGR239C were assigned the names ScPEX18 and ScPEX21 , respectively, and their gene products are referred to as ScPex18p and ScPex21p. PEX18 was PCR amplified from W303 (wild-type) genomic DNA with primers that map to the extreme 5 Ј end of the PEX18 coding region (5 Ј -GGATCCAAACAATGAATAGTAAC-3 Ј ; initiation ATG is underlined) and 49-28 nucleotides 3 Ј to the termination codon (5 Ј -GTTATA-TATCTGAAATTCATGG-3 Ј ). The PCR product was cloned, sequenced, and subcloned under control of the POX1 promoter of the acyl-CoA oxi- ## Table i. yeast strains used in this study ## W303 MATa : ⌬ , gal80 ⌬ , GAL2-ADE2, LYS2::GAL1-HIS3, met2::GAL7-lacz PJ69-4A ⌬ pex7 ⌬ , gal80 ⌬ , GAL2-ADE2, LYS2::GAL1-HIS3, met2::GAL7-lacz, pex7::HIS3 PJ69-4A ⌬ pot1 ⌬ , gal80 ⌬ , GAL2-ADE2, LYS2::GAL1-HIS3, met2::GAL7-lacz, pot1::HIS3 PJ69-4A ⌬ pex18 ⌬ pex21 ⌬ , gal80 ⌬ , GAL2-ADE2, LYS2::GAL1-HIS3, met2::GAL7-lacz, pex18::TRP1, pex21::URA3 PJ69-4A ⌬ pex13 ⌬ , gal80 ⌬ , GAL2-ADE2, LYS2::GAL1-HIS3, met2::GAL7-lacz, pex13::URA3 PJ69-4A ⌬ pex14 ⌬ , gal80 ⌬ , GAL2-ADE2, LYS2::GAL1-HIS3, met2::GAL7-lacz, pex14::URA3 *For expression of URA3 based plasmids in W303 ⌬ pex18 ⌬ pex21 , the URA3 gene integrated at the PEX21 locus was first disrupted with HIS3 , using the marker-swap plasmid pUH4 [bib_ref] Marker swap" plasmids: convenient tools for budding yeast molecular genetics, Cross [/bib_ref]. [formula] :URA3 W303 ⌬ pex18 ⌬ pex21 * MATa , [/formula] dase gene into plasmids pRS315 [bib_ref] A system of shuttle vectors and yeast host strains designed for efficient..., Sikorski [/bib_ref] and Yep351 [bib_ref] Yeast/E. coli shuttle vectors with multiple unique restriction sites, Hill [/bib_ref] to produce pYcp-PEX18 and pYep-PEX18, respectively. PEX21 was amplified in a similar way, using PCR primers mapping to the extreme 5 Ј end of the PEX21 coding region (5 Ј-TAGAATACCATGC-CCAGTGTCT-3Ј; initiation ATG is underlined) and 397-378 nucleotides 3Ј to the stop codon (5Ј-AGCAAGCAACACCAAAACCA-3Ј) and cloned to produce pYcp-PEX21 and pYep-PEX21. Epitope tagging of PEX18 and PEX21 was achieved by replacing the downstream PCR primers with primers designed to extend the reading frames of PEX18 (5Ј-TGG-GACGTCGTATGGGTAAGCAATTCTGTCTTCAACATC-3Ј) and PEX21 (5Ј-TGGGACGTCGTATGGGTAATCAAGTATGTCTTTGT-GAAT-3Ј) with a partial copy of the sequence encoding the hemagglutinin nonapeptide epitope (YPYDVPDYA), incorporating an AatII restriction site positioned to allow subsequent in-frame cloning of an AatII fragment encoding the remaining portion of the triple HA epitope tag. Cloning into yeast expression vectors was as for the untagged genes. His 6tagged PEX18 and PEX21 bacterial overexpression constructs were generated by subcloning the two-hybrid library clone inserts encoding the COOH-terminal 176 residues of Pex18p (as a BamHI-BglII fragment) into BamHI-digested pPET15b (to create pPET15b-PEX18), and the COOH-terminal 96 residues of Pex21p (as a SmaI-SmaI fragment) into pPET15b digested with XhoI and filled in with Klenow DNA Polymerase and deoxynucleotides (to create pPET15b-PEX21). For gene disruption, pPEX18::TRP1 was generated by replacement of an EcoRI-EcoRI fragment of PEX18 (coding nucleotides 119-797) with an EcoRI-EcoRI fragment carrying the TRP1 gene. pPEX21::URA3 was prepared by replacement of an EcoRI-NsiI fragment (coding nucleotides 363-end) of PEX21 with an EcoRI-PstI fragment carrying the URA3 gene. A BamHI-SalI fragment of pPEX18::TRP1 and a HindIII-EcoRI (partial) fragment of pPEX21::URA3 were purified after digestion, and used for transformation, individually or consecutively of W303 and PJ69-4A. pPEX7::HIS3, for the disruption of PEX7 from PJ69-4A, was made by replacing a BglII-BglII portion of PEX7, which includes the entire open reading frame, with a BamHI-BamHI fragment containing HIS3. pPOT1:: HIS3, for the disruption of thiolase from PJ69-4A and W303, was made by replacing an SphI-StuI portion of POT1, which includes the promoter and first 776 coding nucleotides, with an SphI-SmaI fragment containing HIS3. pPEX13::URA3 and pPEX14::URA3, for the disruption of PEX13 and PEX14 from PJ69-4A, were made by replacement of a KpnI-SalI fragment of PEX13 (encoding residues 69-145) and a BclI-XhoI fragment of PEX14 (encoding residues 140-201) with URA3-containing KpnI-SalI and BamHI-SalI fragments, respectively. All disruptions were confirmed by PCR on genomic DNA. For construction of the two-hybrid bait plasmid pGBT9-PEX7, an NcoI site was introduced around the PEX7 initiation codon by PCR, and the entire PEX7 coding region was then purified as an NcoI(filled in)-BamHI(partial) digestion product, which was then cloned into the SmaI-BamHI sites of pGBT9 to produce an in-frame fusion with the Gal4p DNA-binding domain. pGBT9-thio (complete thiolase coding sequence) and pGBT9-⌬thio (thiolase coding sequence lacking amino acids 1-16, which constitute the PTS2) were generated by subcloning the appropriate regions of thiolase into pGBT9. Plasmid pChAT-KANL, encoding a version of bacterial chloramphenicol acetyltransferase appended at the COOH terminus with ϪKANL, which is targeted to yeast peroxisomes in a Pex5p-dependent fashion, has been described previously [bib_ref] Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal..., Purdue [/bib_ref] , as have the plasmids encoding epitope-tagged Pex7p at high (pBXNPEB1-HA3 in [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] , now referred to as pBXNPEX7-HA3) and low (YipPEB1-HA3 in [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] , now referred to as YipPEX7-HA3) expression levels, and the plasmid encoding a fusion between the NH 2 -terminal 16 amino acid PTS2 of S. cerevisiae thiolase and GFP (PTS2-GFP; [bib_ref] Targeting of green fluorescent protein to peroxisomes and peroxisome membranes in S...., Huang [/bib_ref]. The plasmid encoding acyl-CoA oxidase fused to GFP was constructed by cloning the POX1 gene (complete with its promoter) into the episomal plasmid Yep351 [bib_ref] Yeast/E. coli shuttle vectors with multiple unique restriction sites, Hill [/bib_ref] , followed by replacement of the termination codon with an AatII restriction site by site-directed mutagenesis, digestion with AatII, bluntending, and in-frame cloning of a blunt-ended XhoI-SacI fragment containing GFP. ## Bacterial overexpression and affinity chromatography E. coli BL21(DE3) cells were transformed with plasmids pPET15b-PEX18 and pPET15b-PEX21, grown to an OD 600 of 0.8, and then grown for an additional three hours in the presence of 1 mM IPTG. Cells were then harvested, resuspended in Binding Buffer (5 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), sonicated, and centrifuged at 14,000 g for 10 min at 4ЊC. The resultant supernatant was loaded onto His- Bind Resin columns (Novagen Inc., Madison, WI), which were then washed extensively before recovery of bound proteins in elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9), in accordance with the manufacturer's recommendations. To test the binding of Pex7p to Pex18p/Pex21p, yeast cells expressing epitope tagged Pex7p were homogenized [bib_ref] Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into..., Thieringer [/bib_ref] in Binding Buffer (containing protease inhibitors) and loaded at a concentration of 5 mg protein/100 l column bed volume onto His- Bind Resin columns that had been preloaded with either Pex18p, Pex21p, or no bait, and extensively washed. The unbound yeast proteins were recovered, and the columns were then rewashed and eluted as usual. Equivalent proportions of the unbound and eluted protein fractions were then analyzed by SDS-polyacrylamide gel electrophoresis followed by either Coomassie blue staining or immunoblotting. ## Miscellaneous Standard techniques of yeast geneticsand molecular biologywere used throughout. Cellular fractionation, density gradient centrifugation, preparation of glass-bead homogenates, ECL immunoblotting, immunoprecipitation, and immunofluorescence were as described previously [bib_ref] Peroxisomes in Saccharomyces cerevisiae: immunofluorescence analysis and import of catalase A into..., Thieringer [/bib_ref] [bib_ref] Peb1p (Pas7p) is an intraperoxisomal receptor for the NH 2 -terminal, type..., Zhang [/bib_ref] [bib_ref] Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal..., Purdue [/bib_ref]. Protease protection assays using Proteinase K-agarose beads were as described [bib_ref] Peb1p (Pas7p) is an intraperoxisomal receptor for the NH 2 -terminal, type..., Zhang [/bib_ref] , except that incubations were at 4ЊC and were performed on purified peroxisomes as well as crude organellar pellets. # Results ## Pex18p and pex21p, novel peroxins capable of interacting with scpex7p: two-hybrid analysis The two-hybrid host strain PJ69-4A and yeast genomic libraries representing all three reading frames, cloned in derivatives of the two-hybrid Gal4p transactivation domain plasmid pGAD424, were generously supplied by Dr. Philip James (University of Wisconsin; [bib_ref] Genomic libraries and a host strain designed for highly efficient two-hybrid selection..., James [/bib_ref]. PJ69-4A represents an improved strain for twohybrid screens, in that it contains three distinct inducible markers, each regulated by a different Gal4p-responsive promoter (see Materials and Methods). We and others have found the GAL2-ADE2 marker to be the most stringent, giving very few (if any) false positives, whereas the GAL1-HIS3 and GAL7-lacz markers are more sensitive (see [fig_ref] Figure 1: Pex18p and Pex21p interact with Pex7p [/fig_ref] but also give many more false positives. Therefore, it was determined that library screening should be carried out on adenine omission plates, with positives subsequently being screened for HIS3 and lacz expression. To screen for Pex7p interacting proteins, the full-length ScPEX7 coding sequence [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] was cloned as an in-frame fusion with the Gal4p DNA-binding domain in plasmid pGBT9 (Clontech Laboratories, Palo Alto, CA) to create the "bait" plasmid pGBT9-PEX7. To validate the usefulness of this bait in PJ69-4A, it was initially tested for interaction with thiolase, which has previously been shown to interact with Pex7p in two-hybrid analyses [bib_ref] The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces..., Rehling [/bib_ref] [bib_ref] Peb1p (Pas7p) is an intraperoxisomal receptor for the NH 2 -terminal, type..., Zhang [/bib_ref]. As expected, interaction of bait with full-length thiolase resulted in an Ade ϩ , His ϩ , ␤-gal ϩ phenotype [fig_ref] Figure 1: Pex18p and Pex21p interact with Pex7p [/fig_ref]. Deletion of the thiolase PTS2 (amino acids 1-16) abrogated activation of all three of these markers. For screening, PJ69-4A was transformed first with pGBT9-PEX7, and then with the transactivation domain libraries. 2 ϫ 10 6 transformants were ob-tained for each library, and these were plated directly onto adenine omission plates. A total of 11 Ade ϩ colonies were identified, all of which were then shown also to display activation of the HIS3 and ␤-galactosidase genes. The library plasmids were rescued from these Ade ϩ clones, propagated in bacteria, purified and retransformed into PJ69-4A with and without bait, to confirm that interaction was dependent upon the simultaneous presence of both fusions. The library plasmids were sequenced, revealing that three different yeast genes, each encoding a protein of unknown function, were represented amongst the eleven positive clones. One of these genes, represented by a single positive, will be discussed in a later communication. 6 of the other 10 positive clones were for gene YHR160C, which encodes a protein of 283 amino acids, and included fusions between the Gal4p transactivation domain and the COOH-terminal 176 (5 clones) or 200 (1 clone) residues. The remaining four clones were for gene YGR239C, which encodes a protein of 288 amino acids, and included fusion between the Gal4p transactivation domain and the COOH-terminal 134 (3 clones) or 96 (1 clone) residues. Based on the data presented in this paper regarding the involvement of these genes in peroxisomal biogenesis, YHR160C and YGR239C will be referred to hereafter as PEX18 and PEX21, respectively. The interactions between Pex7p bait and one library clone for each of Pex18p and Pex21p are illustrated in [fig_ref] Figure 1: Pex18p and Pex21p interact with Pex7p [/fig_ref]. Since Pex7p has previously been shown to interact not only with the PTS2 of thiolase [bib_ref] The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces..., Rehling [/bib_ref] [bib_ref] Peb1p (Pas7p) is an intraperoxisomal receptor for the NH 2 -terminal, type..., Zhang [/bib_ref] [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref] , but also with the peroxins Pex13p [bib_ref] Involvement of Pex13p in PTS2-dependent peroxisomal protein import, Girzalsky [/bib_ref] and Pex14p [bib_ref] Pex14p, a peroxisomal membrane protein binding both receptors of the two PTS-dependent..., Albertini [/bib_ref] [bib_ref] Pex14p is a member of the protein linkage map of Pex5p, Brocard [/bib_ref] [bib_ref] Peroxisomes: organelles at the crossroads, Erdmann [/bib_ref] , we extended our two-hybrid analysis to ask whether these proteins may have an involvement in the observed interaction between Pex7p and Pex18p/Pex21p. To do this, the genes encoding thiolase, Pex13p, and Pex14p, were each deleted from the two-hybrid host PJ69-4A to create strains PJ69-4A⌬pot1, PJ69-4A⌬pex13 and PJ69-4A⌬pex14, respectively, and the Pex7p-Pex18p/Pex21p interactions were reassessed in these disruption strains. As shown in [fig_ref] Figure 1: Pex18p and Pex21p interact with Pex7p [/fig_ref] , none of these deletions had any observable effect on the Pex7p-Pex18p/Pex21p interactions. ## In vitro binding To complement the in vivo two-hybrid interaction analyses, we sought to investigate the ability of Pex7p to bind Pex18p and Pex21p in vitro. To achieve this, the COOHterminal regions of Pex18p and Pex21p that interact with Pex7p in the two-hybrid system were overexpressed as polyhistidine (His 6 ) tagged proteins in bacteria, and subsequently purified on polyhistidine affinity columns. The His 6 -tagged proteins were specifically retained on the columns, to which were then added protein extracts from yeast cells expressing hemagglutinin epitope-tagged Pex7p. Coomassie blue staining [fig_ref] Figure 2: Pex18p and Pex21p interact with Pex7p in vitro [/fig_ref] revealed that almost all of the yeast proteins failed to bind to the columns to any great extent, and none showed any discernible difference in binding to columns loaded with Pex18p, Pex21p, or neither. However, as shown in [fig_ref] Figure 2: Pex18p and Pex21p interact with Pex7p in vitro [/fig_ref] B, immunoblot analysis revealed that the epitope-tagged Pex7p was specifically retained on the Pex18p and Pex21p columns, but not on columns lacking either of these peroxins. This supports the two-hybrid data indicating that Pex18p and Pex21p can specifically interact with Pex7p. ## Pex18p and pex21p are weakly homologous to each other Sequence comparison of Pex18p and Pex21p revealed a weak but significant level of homology [fig_ref] Figure 3: Bestfit alignment of the protein sequences of Pex18p and Pex21p [/fig_ref] , raising the possibility that the ability of both these proteins to interact with Pex7p may result from their sharing a common structural domain. Whereas the observed homology is distributed throughout the protein sequences (23% identity overall), it is most prominent at the COOH termini (35% identity in the last 60 amino acids). This conserved COOHterminal domain most likely represents the region of Pex18p/ Pex21p responsible for interaction with Pex7p, since it is present in all the PEX18 and PEX21 library clones isolated with the PEX7 bait, and the shortest interacting clone contains only the last 96 amino acids of Pex21p. in the yeast two-hybrid assay. Host strain PJ69-4A was transformed with two plasmids, one encoding the Gal4p transactivation domain (TA) alone (Ϫ) or fused to either residues 108-283 of Pex18p (Pex18), residues 155-288 of Pex21p (Pex21), or full-length thiolase (Thio), and the other encoding the DNA binding domain (BD) of Gal4p fused to full-length Pex7p (Pex7), full-length thiolase (Thio) or thiolase lacking the NH 2 -terminal 16 amino acids (Thio⌬1-16). Transformants were tested for growth on synthetic complete medium with (ϩAde ϩHis) or without adenine (ϪAde) or histidine (ϪHis). Total cell homogenates were also prepared, and assayed for ␤-galactosidase activity (␤-Gal), which is expressed in nmol/min/mg protein. Ϫ ϭ Ͻ0.5 units of activity. Some experiments were performed using PJ69-4A from which the PEX7, POT1, PEX13, PEX14, or PEX18/PEX21 genes had been deleted (PJ69-4A⌬pex7, PJ69-4A⌬pot1, PJ69-4A⌬pex13, PJ69-4A⌬pex14, and PJ69-4A⌬pex18⌬pex21, respectively). PJ69-4A⌬pex7 and PJ69-4A⌬pot1 transformants were not tested for activation of the GAL1-HIS3 marker, since these strains had been converted to His ϩ during gene disruption. ## Pex18p and pex21p constitute a peroxin family essential for pts2 targeting and growth on oleate medium To investigate the roles of Pex18p and Pex21p in peroxisomal biogenesis, the genes encoding these proteins were disrupted, both individually and together, from wild-type strain W303. The disruption strains were initially tested for growth on plates containing oleate as sole carbon source, which will support cell growth only if peroxisomal ␤-oxidation is functional. Of the single gene disruptions, W303 ⌬pex21 grew at a rate indistinguishable from wild-type, whereas W303⌬pex18 cells grew somewhat more slowly [fig_ref] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished... [/fig_ref]. However, the double disruption strain (W303⌬pex18⌬pex21) showed no growth whatsoever on this medium [fig_ref] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished... [/fig_ref]. To investigate the biochemical basis for this growth defect, we next looked at the subcellular distribution of thiolase, since this is a key enzyme of peroxisomal ␤-oxidation, and is targeted to peroxisomes by the Pex7p-dependent PTS2 targeting pathway [bib_ref] PAS7 encodes a novel yeast member of the WD-40 protein family essential..., Marzioch [/bib_ref] [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref]. Both by indirect immunofluorescence and subcellular fractionation analyses, peroxisomal packaging of thiolase appeared unperturbed in W303⌬pex21 [fig_ref] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished... [/fig_ref]. However, in strain W303⌬pex18 ‫%05ف‬ of thiolase was mistargeted to the cytosol, as assessed by fractionation, and fluorescence was distributed throughout the cytosol (presumably masking the signal from the residual peroxisomal thiolase). Finally, W303⌬pex18⌬pex21 showed complete redistribution of thiolase to the cytosol [fig_ref] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished... [/fig_ref]. These results strongly indicated that Pex18p and Pex21p were specifically involved in PTS2 targeting. To test this further, we investigated the subcellular distribution in these strains of a plasmid encoded fusion between the PTS2 of S. cerevisiae thiolase (amino acids 1-16) and GFP (PTS2-GFP), under the regulation of the thiolase promoter. The targeting of this fusion protein closely resembled that of thiolase, with predominantly peroxisomal fluorescence seen in wild-type and W303⌬pex21, but cytosolic fluorescence in W303⌬pex18 and W303⌬pex18⌬pex21 [fig_ref] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished... [/fig_ref]. Whereas no punctate fluorescence at all was detected in W303⌬pex18⌬pex21, some W303⌬pex21 cells showed a mixture of cytosolic and punctate labeling, consistent with the partial thiolase mistargeting phenotype of this strain. ## Targeting by pts1 or the pts of acyl-coa oxidase do not require pex18p/pex21p To determine whether the involvement of Pex18p/Pex21p in peroxisomal biogenesis extends beyond the PTS2 branch of peroxisomal biogenesis, the disruption strains were assessed for packaging of other peroxisomal proteins. These studies revealed that targeting via the PTS1 pathway, as represented by the multi-functional ␤-oxidation protein (ends ϪSKL; [bib_ref] Peroxisomal multifunctional beta-oxidation protein of Saccharomyces cerevisiae. Molecular analysis of the FOX2..., Hiltunen [/bib_ref] , catalase A (ends ϪSKF; [bib_ref] Two independent peroxisomal targeting signals in catalase A of Saccharomyces cerevisiae, Kragler [/bib_ref] and a plasmid-encoded fusion between ChAT and the human catalase PTS1 (ϪKANL; [bib_ref] Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal..., Purdue [/bib_ref] , was not affected by the single or double disruptions [fig_ref] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished... [/fig_ref]. Similarly, targeting of acyl-CoA oxidase (AOX), which is believed to contain a yet-to-be-defined PTS3 signal (since it lacks both PTS1 and PTS2; [bib_ref] Structure and transcriptional control of the Saccharomyces cerevisiae POX1 gene encoding acyl-coenzyme..., Dmochowska [/bib_ref] , was normal in all strains, as assessed by fluorescence analysis of cells expressing a fusion between AOX and GFP which is targeted efficiently to peroxisomes (Skoneczny, M., P.E. Purdue, and P.B. Lazarow, manuscript in preparation). (Pex21), or neither (Ϫ) were first applied to His 6 -affinity columns, which were then loaded with glass bead homogenates of yeast expressing epitope tagged Pex7p (plasmid pBXNPEB1-HA3 in [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] , now referred to as pBXNPEX7-HA3). The unbound yeast proteins were recovered and, after extensive washing, the bound proteins were eluted together with the His 6 -tagged Pex18p/Pex21p. Aliquots of the unbound and bound fractions were resolved in duplicate by SDS PAGE, and subjected to (A) Coomassie blue staining, which detected the eluted Pex18p (which migrates as a dimer) and Pex21p, and (B) immunoblotting with monoclonal antibody 12CA5 to detect the epitope-tagged Pex7p. An abundant unidentified yeast protein that adheres to the columns even in the absence of His 6 -tagged bait is marked by an asterisk. Cells were grown to mid-log phase in liquid synthetic complete medium, and then applied at various dilutions (10 5 , 10 4 , or 10 3 cells per spot) to plates containing synthetic complete medium with either oleic acid (SC-OLEATE) or dextrose (SC-DEXTROSE) as sole carbon source, and grown at 30ЊC. The bottom two panels show the double knockout strain transformed with centromeric plasmids encoding epitope tagged Pex18p (pYcp-PEX18) or Pex21p (pYcp-PEX21). (B) Growth in liquid oleate media. Cells were precultured to mid-log phase in SCD medium and then inoculated at a density of 10 5 cells/ml into liquid SCO. (C) Fluorescence and cell fractionation. Wild-type cells, and cells lacking Pex18p and/ or Pex21p, were grown for peroxisome induction, and analyzed by immunofluorescence with antibodies against thiolase (top row) and catalase A (third row). These same strains were transformed with plasmids expressing chloramphenicol acetyl transferase (ChAT) appended with the PTS1 ϪKANL [bib_ref] Targeting of human catalase to peroxisomes is dependent upon a novel COOH-terminal..., Purdue [/bib_ref] , or a fusion between the thiolase PTS2 and GFP (PTS2-GFP; [bib_ref] Targeting of green fluorescent protein to peroxisomes and peroxisome membranes in S...., Huang [/bib_ref] , or a fusion between acyl-CoA oxidase and GFP (AOX-GFP), and analyzed with anti-ChAT antibodies (fourth row), or by GFP autofluorescence (second and fifth rows). Induction was with YPGO in all cases except for PTS2-GFP fluorescence, when SCEO was used. For fractionation, cells were grown for peroxisome induction in YPGO, and fractionated into postnuclear pellet (P) and supernatant (S) fractions, which were run in equivalent proportions on SDS-polyacrylamide gels, and immunoblotted to detect thiolase and the ␤-oxidation multifunctional protein (MFP). All the strains are in a W303 genetic background. ## Pex18p/pex21p are required for peroxisomal targeting of epitope-tagged pex7p Reports from several laboratories have concluded that yeast Pex7p is associated, at least in part, with peroxi-somes [bib_ref] PAS7 encodes a novel yeast member of the WD-40 protein family essential..., Marzioch [/bib_ref] [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref]. However, observations conflict over whether this protein is an exclusively intra-peroxisomal import receptor, or has a substantial presence and function in the cytosol. Data from our laboratory indicate that, in S. cerevisiae, Pex7p tagged at the COOH terminus with three repeats of the hemagglutinin nonapeptide recognized by monoclonal antibody 12CA5 (Pex7p-HA3) is, within experimental error, confined to the peroxisomal matrix when expressed at wild-type levels, but also accumulates in the cytosol when overexpressed [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref]. Since these results suggest that Pex7p may function within the peroxisomal matrix, we proceeded to investigate the distribution of this same epitopetagged Pex7p in the W303⌬pex18 and W303⌬pex18⌬pex21 strains, which show deficient PTS2 targeting. Subcellular fractionation analysis revealed that the peroxisomal localization of Pex7p-HA3 seen in wild-type cells was essentially absent in W303⌬pex18⌬pex21 cells, with only a trace detectable in the organellar pellet [fig_ref] Figure 5: Peroxisomal targeting of Pex7p is somewhat decreased in cells lacking Pex18p, and... [/fig_ref]. This dependence upon Pex18p/Pex21p for the presence of Pex7p in peroxisomes was found not only when Pex7p-HA3 was expressed at wild-type levels [fig_ref] Figure 5: Peroxisomal targeting of Pex7p is somewhat decreased in cells lacking Pex18p, and... [/fig_ref] , but also when it was markedly overexpressed [fig_ref] Figure 5: Peroxisomal targeting of Pex7p is somewhat decreased in cells lacking Pex18p, and... [/fig_ref]. Consistent with the intermediary PTS2 targeting defect seen in W303⌬pex18, this strain showed a level of peroxisomal targeting of Pex7p-HA3 significantly lower than wild-type, but higher than seen in W303⌬pex18⌬pex21 [fig_ref] Figure 5: Peroxisomal targeting of Pex7p is somewhat decreased in cells lacking Pex18p, and... [/fig_ref]. As previously reported, absence of thiolase did not affect the intracellular distribution of Pex7p [fig_ref] Figure 5: Peroxisomal targeting of Pex7p is somewhat decreased in cells lacking Pex18p, and... [/fig_ref]. ## Pex7p can interact with the thiolase pts2 and pex18p simultaneously Since Pex18p and Pex21p participate in the PTS2 peroxisomal targeting pathway, we next tested whether they were capable of two-hybrid interaction with thiolase, which contains an NH 2 -terminal PTS2. To do this, the PEX18 and PEX21 clones previously isolated from the two-hybrid libraries were each cotransformed into PJ69-4A with plasmids encoding full-length thiolase, or thiolase lacking the NH 2 -terminal 16 amino acid PTS2, fused to the Gal4p DNA-binding domain, and plated on media lacking adenine or histidine. Interestingly, full-length thiolase exhibited a clear interaction with Pex18p (Ade ϩ , His ϩ , ␤-gal ϩ ), and a somewhat lesser interaction with Pex21p (His ϩ , ␤-gal ϩ ), whereas truncated thiolase showed no interaction [fig_ref] Figure 1: Pex18p and Pex21p interact with Pex7p [/fig_ref]. These results suggested that Pex18p/Pex21p may be capable of interaction with the PTS2 of thiolase. However, an alternative explanation was suggested by the fact that Pex7p has now been observed to interact with both thiolase [bib_ref] The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces..., Rehling [/bib_ref] [bib_ref] Peb1p (Pas7p) is an intraperoxisomal receptor for the NH 2 -terminal, type..., Zhang [/bib_ref] [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref] and Pex18p/Pex21p, raising the possibility that cellular Pex7p is serving as a bridge between the Pex18p/Pex21p and thiolase fusions. Therefore, these transformations were repeated in PJ69-4A from which the PEX7 gene had been disrupted (PJ69-4A⌬pex7). In this strain, no interactions were detected, suggesting that the Gal4p activation seen in the full-length thiolase cotransformants probably results from simultaneous interaction of cellular Pex7p with both thiolase and Pex18p (or Pex21p) fusions, although the presence of other bridging proteins can not be ruled out at this stage. Consistent with this, the Pex7p-Pex18p/Pex21p interaction was not affected by disruption of the thiolase gene, and the Pex7pthiolase interaction was not altered by disruption of the PEX18 and PEX21 genes [fig_ref] Figure 1: Pex18p and Pex21p interact with Pex7p [/fig_ref]. ## Pex18p localizes to the cytoplasm and outer surface of peroxisomes To localize Pex18p and Pex21p within cells, clones encoding these proteins appended at their COOH terminus with three copies of the hemagglutinin nonapeptide epitope were generated and expressed in yeast cells. Both tagged proteins were capable of rescuing oleate growth [fig_ref] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished... [/fig_ref] and thiolase packaging in W303⌬pex18⌬pex21 when expressed from the oleate-inducible POX1 promoter in low-copy plasmids. Fractionation analysis revealed that Pex18p was largely cytosolic, but also present at low levels (Ͻ10% of the total) in the organellar pellet . This pattern of distribution was also seen when [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] , now referred to as pBXNPEX7-HA3) were fractionated into postnuclear pellet (P) and supernatant (S) fractions, which were then analyzed by SDS-polyacrylamide gels and immunoblotting with monoclonal antibody 12CA5 (to detect the tagged Pex7p) and antibodies against the ␤-oxidation multifunctional protein (MFP). (B) Immunoprecipitation of epitope tagged Pex7p expressed at wild-type levels (plasmid YipPEB1-HA3 in [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] , now referred to as YipPEX7-HA3). Total cell extracts (T) and organellar pellets (P) from YPGO-induced cultures of W303, W303-expressing tagged Pex7p, and W303⌬pex18⌬pex21-expressing tagged Pex7p were separated on SDS-polyacrylamide gels, and immunoblotted with monoclonal antibody 12CA5. (C) Strains overexpressing epitope tagged Pex7p were analyzed by immunofluorescence with 12CA5. tagged Pex18p was expressed in cells lacking either thiolase, Pex7p or Pex14p, a putative Pex7p receptor on the external face of the peroxisomal membrane (results not shown). Increasing expression of tagged Pex18p increased the amount of targeting to the organellar pellet, indicating that targeting is not saturated at the lower expression level. Nycodenz gradient fractionation of the organellar pellet further localized the pelletable Pex18p to peroxisomes, although trace amounts also copurified with mitochondria . Protease protection analysis of the organellar pellet (or peroxisomal peak fractions from the gradient) revealed that almost all of the peroxisomal tagged Pex18p is readily proteolytically digested under conditions in which thiolase (a matrix protein) is only digested in the presence of detergent , although lengthy exposure of the immunoblots did identify a small pool of protected Pex18p. This suggests that peroxisomal Pex18p is primarily located on the external face of the peroxisomal membrane, but that the existence of a small pool of intraperoxisomal Pex18p can not be ruled out. A primarily cytosolic localization of Pex18p was supported by immunofluorescence analysis, which showed fluorescence throughout the cell . A similar immunofluorescence picture was obtained with cells expressing tagged Pex21p , indicating that this protein is also primarily cytosolic, which is not surprising in view of the apparent redundancy of function of Pex18p and Pex21p. However, we were unable to determine whether any Pex21p is organellar, since the tagged Pex21p was highly unstable during cellular fractionation procedures (data not shown). # Discussion Our current model of peroxisome biogenesis describes a branched pathway of matrix protein import, with each branch representing an import receptor specific for one of several distinct classes of PTS, followed by a shared membrane translocation process. At least three branches exist, two representing the well-characterized COOH-terminal PTS1 and NH 2 -terminal PTS2 signals, and the other(s) representing proteins lacking either of these, most notably acyl-CoA oxidase. In recent years, much has been learned about PTS2 targeting through the cloning and characterization from several species of Pex7p, an import receptor . Pex18p and Pex21p are functional when COOH terminally tagged with HA epitopes, and localize primarily to the cytosol. Pex18p is partially peroxisomal. (A) Cells lacking both Pex18p and Pex21p (⌬pex18⌬pex21)before and after transformation with low copy (pYcp-PEX18, pYcp-PEX21) or high copy (pYep-PEX18, pYep-PEX21) plasmids encoding HA epitopetagged Pex18p or Pex21p were analyzed by immunofluorescence with anti-thiolase (top panels) and 12CA5 (detects the HAtagged proteins; bottom panels). (B) Wild-type cells expressing epitope-tagged Pex18p from low copy (pYcp-PEX18) and high copy (pYep-PEX18) plasmids were fractionated into organellar pellet (P) and supernatant (S) fractions, which were run in equivalent proportions on SDS-polyacrylamide gels, and immunoblotted with monoclonal 12CA5 to detect Pex18p (top), and anti-thiolase (bottom). (C) The organellar pellet from wild-type W303 cells transformed with plasmid pYep-PEX18 was further fractionated by Nycodenz gradient centrifugation. Fractions were run on a polyacrylamide gel and immunoblotted to detect Pex18p (monoclonal 12CA5), and to detect thiolase, ADP/ATP carrier and Kar2p, which are markers for the peroxisomes, mitochondria and endoplasmic reticulum, respectively. The fractions are labeled from top to bottom of the gradient. which interacts with PTS2 sequences and is required for peroxisomal targeting of proteins bearing these signals [bib_ref] PAS7 encodes a novel yeast member of the WD-40 protein family essential..., Marzioch [/bib_ref] [bib_ref] The import receptor for the peroxisomal targeting signal 2 (PTS2) in Saccharomyces..., Rehling [/bib_ref] [bib_ref] Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic..., Braverman [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a..., Motley [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue..., Purdue [/bib_ref] [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref]. In this paper, we report the identification and initial characterization of Pex18p and Pex21p, two novel S. cerevisiae peroxins required for PTS2 targeting. Substantial evidence indicates that Pex18p and Pex21p are specifically involved in the PTS2, and no other, branch of peroxisomal protein biogenesis. Most importantly, peroxisomal import of PTS2-targeted thiolase, or of a fusion protein consisting of the yeast thiolase PTS2 (amino acids 1-16) fused to GFP, is completely abolished in cells lacking Pex18p and Pex21p, whereas targeting of PTS1 proteins, and of acyl-CoA oxidase (which has neither PTS2 nor PTS1) fused to GFP, are normal. In addition, Pex18p and Pex21p each interact with Pex7p both in two-hybrid assays and in vitro. Pex7p has been shown to function as a PTS2 receptor, not only in yeast, but also in humans, where its deficiency causes rhizomelic chondrodysplasia punctata (RCDP; [bib_ref] Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic..., Braverman [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is a peroxisomal protein targeting disease caused by a..., Motley [/bib_ref] [bib_ref] Rhizomelic chondrodysplasia punctata is caused by deficiency of human PEX7, a homologue..., Purdue [/bib_ref]. Although thiolase is the only currently known yeast PTS2 protein, other human PTS2 proteins have been identified and shown to be deficient in RCDP [bib_ref] Nucleotide sequence of human alkyl-dihydroxyacetonephosphate synthase cDNA reveals the presence of a..., Vet [/bib_ref] [bib_ref] Identification of PAHX, a Refsum disease gene, Mihalik [/bib_ref] [bib_ref] Refsum disease is caused by mutations in the phytanoyl-CoA hydroxylase gene, Jansen [/bib_ref] , indicating that Pex7p (and, by extension, Pex18p and Pex21p) mediate targeting not just of thiolase, but of PTS2s in general. It is considerable interest to note that whereas the molecular basis of PTS2 targeting pathway is conserved between S. cerevisiae and humans, current evidence indicates that this may not be the case for Yarrowia lipolytica. In this latter yeast, no orthologue for Pex7p has yet been identified, but a recent report describes a novel peroxin, Pex20p, required for thiolase import [bib_ref] Pex20p of the yeast Yarrowia lipolytica is required for the oligomerization of..., Titorenko [/bib_ref]. YlPex20p shows no obvious similarity in molecular function to either Pex7p or to the Pex18p/Pex21p peroxins described in this paper. Instead, it functions by binding cytosolic thiolase in a PTS2-independent manner, and mediating the subsequent oligomerization and biogenesis of thiolase. Proteome-wide searches with the Y. lipolytica Pex20p sequence fail to identify any significant candidates for a S. cerevisiae Pex20p orthologue and, while it is too early to exclude the possible existence of ScPex20p, the weight of evidence presently available tends to suggest that the molecular machinery for PTS2/thiolase targeting in S. cerevisiae (and humans) is substantially different from that found in Y. lipolytica. A number of observations indicate that Pex18p and Pex21p constitute a two-member family of peroxins of related and redundant function. First, as mentioned above, both of these proteins were identified by interaction with the same bait, Pex7p, in a two-hybrid screen, and they both bind epitope-tagged Pex7p in vitro. Second, they are similar in size and display significant sequence homology to each other, but show no striking similarity to any other S. cerevisiae proteins. This homology is distributed throughout their entire lengths, but is particularly noteworthy in the COOH-terminal domain common to all the clones isolated by virtue of interaction with Pex7p. Third, presence of either Pex18p or Pex21p, but not the other, is sufficient to support substantial levels of PTS2 targeting, with abolition of this pathway only occurring when both these proteins are absent. Although Pex18p and Pex21p share functional redundancy, they appear to make quantitatively different contributions to PTS2 targeting. For example, peroxisomal targeting of thiolase is indistinguishable from wild-type in cells lacking Pex21p, whereas cell lacking Pex18p mislocalize ‫%05ف‬ of thiolase to the cytosol. Although the respective contributions of Pex18p and Pex21p to PTS2 targeting in wild-type cells have not been established, these data indicate that Pex18p may bear most of the burden. The level of thiolase targeting in ⌬pex18 cells (and ⌬pex21 cells) is sufficient to support growth on oleic acid as sole carbon source, which requires functional peroxisomal ␤-oxidation. This provides an explanation as to why pex18 and pex21 mutants have not been identified by genetic screens for pex mutants unable to use oleic acid. It is, however, interesting to note that ⌬pex18 cells grow more slowly than wild-type (or ⌬pex21) cells on oleic acid, suggesting that levels of peroxisomal thiolase may be rate limiting for ␤-oxidation, although the existence of yet-to-be-identified additional PTS2-targeted proteins required for peroxisomal ␤-oxidation can not be ruled out [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref]. What might the function(s) of Pex18p and Pex21p be? Data presented in this paper show that peroxisomal targeting of Pex7p is virtually abolished in a strain lacking these peroxins, suggesting that their primary function may be associated with the biogenesis of Pex7p. However, interpretation of this data is complicated by the controversy that surrounds the localization and function of Pex7p. We have previously reported that in S. cerevisiae, Pex7p COOH terminally tagged with HA epitopes is both functional and exclusively peroxisomal (within experimental error) when at wild-type PEX7 expression levels [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref]. In contrast, a predominantly cytosolic localization of the S. cerevisiae protein was reported by one group, based on overexpression of Pex7p appended NH 2 -terminally with a c-myc tag [bib_ref] PAS7 encodes a novel yeast member of the WD-40 protein family essential..., Marzioch [/bib_ref]. In a different yeast, P. pastoris, Pex7p was localized to both the peroxisomal matrix and the cytosol [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref]. In human cells, we have found that overexpressed . Protease protection analysis shows that peroxisomal Pex18p is primarily located on the external face of the peroxisomal membrane. An organellar fraction of wild-type cells overexpressing HA-tagged Pex18p was digested with a final concentration of 5 g/ml Proteinase K-agarose in the presence and absence of 0.1% Triton X-100. Reactions were stopped with 1 mM PMSF after various times, and the samples were separated by SDS-PAGE, and immunoblotted to detect thiolase, a known matrix protein, and the tagged Pex18p. A proteolytic thiolase digestion product is indicated by an asterisk. Pex7p is partly peroxisomal and partly cytosolic , whereas others report an exclusively cytosolic localization [bib_ref] Human PEX7 encodes the peroxisomal PTS2 receptor and is responsible for rhizomelic..., Braverman [/bib_ref]. This range of localization results has spawned a variety of hypotheses regarding Pex7p function. Whereas we have proposed that a peroxisomal matrix localization suggests that Pex7p may function as an intraperoxisomal receptor [bib_ref] PEB1 (PAS7) in Saccharomyces cerevisiae encodes a hydrophilic, intraperoxisomal protein which is..., Zhang [/bib_ref] , others have proposed models in which Pex7p may act as a mobile receptor, moving (with PTS2 cargo) from cytosol to either the peroxisomal outer surface, or the peroxisomal matrix, delivering cargo, and recycling for additional rounds of import [bib_ref] PAS7 encodes a novel yeast member of the WD-40 protein family essential..., Marzioch [/bib_ref] [bib_ref] Peroxisomes: organelles at the crossroads, Erdmann [/bib_ref] [bib_ref] A mobile PTS2 receptor for peroxisomal protein import in Pichia pastoris, Elgersma [/bib_ref]. Interestingly, in spite of the fundamental differences between these various models, they all stipulate a requirement for Pex7p to move from cytosol to peroxisome, and can therefore all accommodate in principle Pex18p/Pex21p as factors required for this targeting event. Since Pex18p and Pex21p both interact with Pex7p, we can now envisage this pair of peroxins acting as a receptor(s) for targeting of Pex7p from the cytosol to peroxisomes, and that loss of PTS2 targeting in ⌬pex18 ⌬pex21 cells results from Pex7p failing to reach its site of action, the peroxisomal matrix (according to the intraperoxisomal receptor model), or failing to cycle between cytosol and peroxisome (according to the mobile receptor model). Localization of Pex18p to the cytosol and peroxisomal surface is consistent with a role for this protein in ferrying Pex7p to the peroxisomal membrane translocation machinery. Pex14p and Pex13p have been identified as peroxisomal membrane proteins capable of interacting with Pex7p [bib_ref] Pex14p, a peroxisomal membrane protein binding both receptors of the two PTS-dependent..., Albertini [/bib_ref] [bib_ref] Pex14p is a member of the protein linkage map of Pex5p, Brocard [/bib_ref] [bib_ref] Peroxisomes: organelles at the crossroads, Erdmann [/bib_ref] [bib_ref] Involvement of Pex13p in PTS2-dependent peroxisomal protein import, Girzalsky [/bib_ref]. These peroxins are not directly involved in the Pex18p-Pex7p and Pex21p-Pex7p interactions, as evidenced by the maintenance of these interactions in two-hybrid strains lacking Pex13p or Pex14p, but they are required for PTS2 targeting, raising the possibility that Pex7p is transferred from Pex18p/ Pex21p to Pex14p and/or Pex13p at the outer face of the membrane. The mobile receptor model suggests that interaction of Pex7p with Pex14p/Pex13p at the external face of the peroxisomal membrane may correspond to a docking event, before recycling of Pex7p to the cytosol, but it seems equally likely that Pex14p/Pex13p may be part of the translocation machinery required for import of Pex7p. It remains to be seen whether yet more peroxins are involved in the PTS2 pathway and the biogenesis of Pex7p. Although all three previously known Pex7p-interacting proteins (thiolase, Pex13p and Pex14p) are dispensable for the Pex7p-Pex18p and Pex7p-Pex21p interactions, it remains a possibility that other, as yet unidentified, peroxins are involved in mediating these interactions. It is curious to note that, despite screening sufficient library clones with Pex7p bait to cover the entire genome several times over, we did not isolate any interacting library clones for Pex14p, Pex13p, or for any PTS2 proteins, including thiolase. To some extent, this can be accounted for by the intrinsic underrepresentation or absence in twohybrid libraries of some 5Ј coding regions, due to the presence of upstream in-frame termination codons close to translational initiation codons [bib_ref] Genomic libraries and a host strain designed for highly efficient two-hybrid selection..., James [/bib_ref]. Thus, whereas a transactivation domain plasmid designed to ex-press the extreme NH 2 -terminal thiolase PTS2 does interact with the Pex7p bait, the existence of such a clone in the two-hybrid libraries is legislated against by the presence of a TAA triplet 15-18 bases upstream of the thiolase (and PTS2) initiation codon [bib_ref] A new glucose-repressible gene identified from the analysis of chromatin structure in..., Igual [/bib_ref]. This explains why thiolase was not identified in our screens, and suggests that the failure to detect other proteins with NH 2 -terminal PTS2s in our library screens does not necessarily imply that such proteins do not exist, a conclusion consistent with a recent suggestion that Pex11p may be PTS2 targeted [bib_ref] Peroxisome biogenesis: Involvement of ARF and coatomer, Passreiter [/bib_ref]. Since the regions of Pex14p and Pex13p which interact with Pex7p have not been mapped, it is unknown whether the failure to detect these proteins can be accounted for in a similar way. Another interesting observation arising from our two-hybrid analysis is that thiolase, fused to the Gal4p DNA-binding domain, interacts with Pex18p (or Pex21p) fused to the transactivation domain, but that this is dependent upon the presence of cellular Pex7p. Disruption of the PEX7 gene from the two-hybrid host strain abolishes this interaction. The most likely explanation of this is that cellular Pex7p, despite its low abundance, can simultaneously bind the thiolase and Pex18p fusions, and thus create a functional Gal4p hybrid, which in turn suggests that the thiolase and Pex18p/ Pex21p binding sites of Pex7p are distinct. A similar scenario has recently been proposed for yeast peroxins Pex5p, Pex7p, Pex14p and Pex17p [bib_ref] Pex17p of Saccharomyces cerevisiae is a novel peroxin and component of the..., Huhse [/bib_ref] [bib_ref] Involvement of Pex13p in PTS2-dependent peroxisomal protein import, Girzalsky [/bib_ref] , where the Pex5p-Pex17p and Pex5p-Pex7p interactions are abolished when PEX14 is disrupted from the two-hybrid host strain. Such findings emphasize that the two-hybrid system can detect indirect, as well as direct, interactions, and while this extends the potential of this type of analysis, it also counsels caution in the interpretation of its results. [fig] Figure 1: Pex18p and Pex21p interact with Pex7p [/fig] [fig] Figure 3: Bestfit alignment of the protein sequences of Pex18p and Pex21p. The arrowheads indicate the fusion junctions between Gal4p transactivation domain and COOH-terminal regions of Pex18p and Pex21p of the clones isolated from twohybrid libraries with Pex7p bait. [/fig] [fig] Figure 2: Pex18p and Pex21p interact with Pex7p in vitro. Bacterial extracts from E. coli BL21 (DE3) cells overexpressing His 6tagged Pex18p (Pex18), Pex21p [/fig] [fig] Figure 4: Growth on oleic acid and packaging of thiolase into peroxisomes are abolished in cells lacking both Pex18p and Pex21p, but are supported fully by Pex18p alone, and partially by Pex21p alone. (A) Growth on plates. [/fig] [fig] Figure 5: Peroxisomal targeting of Pex7p is somewhat decreased in cells lacking Pex18p, and almost abolished in cells lacking both Pex18p and Pex21p. (A) Strains overexpressing epitope tagged Pex7p (plasmid pBXNPEB1-HA3 in [/fig]

Assessment of the Substance Antioxidative Profile by Hyaluronan, Cu(II) and Ascorbate ## Introductory remarks The website "Free Radical School Presentations"serves as "an archive" for many of the Free Radical School Workshop and Virtual School lectures that were organized by the Society for Redox Biology and Medicine and presented at SfRBM's Annual Meeting within 15 years (the first one in 1997 and the last one in 2011). This website is intended to be a source for basic education and reference material for a wide variety of important topics in the field of redox research including the experimental methods to assess the substance antioxidative profile. In the 1990s, the research activities of many scientific teams directly or indirectly focused on the thesis that antioxidants are the "miracle substances" that can effectively intervene in both the acute and chronic phases of many diseases, including aging. Reaction systems for evaluating the antioxidant activity of a substance also contain a generator of oxidant(s) and a probe whose intactness during the reaction proves the antioxidant activity of the substance assessed. In this minireview the primary/initiating oxidants are the OH radicals, the secondary oxidants are alkyloxy-and alkylperoxy-type radicals. The probe is the organism's own macromolecule, namely a high-molar-mass hyaluronan. The kinetics of the hyaluronan oxidative degradation has been most properly monitored by changing the dynamic viscosity of the reaction solution. ## Sequestration of copper cations with ascorbate The biogenic transition metal-copper-is found in the healthy human body as cupric and cuprous cations, whereas 95% of the total copper content is bound to the blood plasma protein-ceruloplasmin. Each ceruloplasmin macromolecule binds eight copper cations, of which two are readily released. The normal level of serum ceruloplasmin concentration is in a range of 1.52-2.65 µmol/L. Thus, under physiological conditions the concentration of loosely bound copper (cupric/cuprous cations) can reach up to ≈0.66 µmol/L. The patho-physiological range of ceruloplasmin blood serum level is significantly increased, since this metalloprotein belongs to the acute phase reactants, the level of which rises during acute and chronic inflammations, infection, trauma, Alzheimer's disease, etc. Ascorbic acid/vitamin C, in the form of ascorbate, is ubiquitous in the human body. The ascorbate molecule (Asc − ) in a living organism functions also as a coordinating low molar mass ligand. In case of ascorbate-iron pair, ascorbate may serve both as a Fe(III)/Fe(II) chelating agent and reductant of Fe(III) to Fe(II) cations. In latter case, under the aerobic conditions ascorbate with ferrous cation forms a coordination complex: ascorbate-Fe(II)-dioxygen. This, the so called Udenfriend oxidative complex/system, is a very efficient oxidative agent used, e.g., by organic chemists to hydroxylate aromatic compounds, saturate hydrocarbons to alcohols, olefins to epoxides, etc.. The paramagnetic bivalent Cu(II) (outermost orbitals = 3d 9 ) represents the most stable oxidation state of copper. Since ascorbate acts as a powerful reducing agent with a standard reduction potential E' - of +0.282 V for the redox couple Asc - − /Asc − at pH 7, it should reduce Cu(II) to Cu(I). Thus, taking into account that the standard reduction potential of the pair Cu(II)/Cu(I) is +0.16 V, Cu(I) should be able to reduce O 2 molecules to yield directly H 2 O 2 . However, as often claimed, the so called Weissberger systemascorbate-Cu(II/Cu(I)-dioxygen-generates hydrogen peroxide molecules (cf. Scheme 1), or due to the decomposition of H 2 O 2 by Cu(I) complex through Fenton type reaction, the Weissberger system became one of the most potent generators of hydroxyl radicals. Pharmaceutics 2021, 13, 1815 2 of 13 disease, etc. Ascorbic acid/vitamin C, in the form of ascorbate, is ubiquitous in the human body. The ascorbate molecule (Asc -) in a living organism functions also as a coordinating low molar mass ligand. In case of ascorbate-iron pair, ascorbate may serve both as a Fe(III)/Fe(II) chelating agent and reductant of Fe(III) to Fe(II) cations. In latter case, under the aerobic conditions ascorbate with ferrous cation forms a coordination complex: ascorbate---Fe(II)---dioxygen. This, the so called Udenfriend oxidative complex/system, is a very efficient oxidative agent used, e.g., by organic chemists to hydroxylate aromatic compounds, saturate hydrocarbons to alcohols, olefins to epoxides, etc.. The paramagnetic bivalent Cu(II) (outermost orbitals = 3d 9 ) represents the most stable oxidation state of copper. Since ascorbate acts as a powerful reducing agent with a standard reduction potential E'° of +0.282 V for the redox couple Asc - − /Asc − at pH 7, it should reduce Cu(II) to Cu(I). Thus, taking into account that the standard reduction potential of the pair Cu(II)/Cu(I) is +0.16 V, Cu(I) should be able to reduce O2 molecules to yield directly H2O2. However, as often claimed, the so called Weissberger system-ascorbate---Cu(II/Cu(I)---dioxygen-generates hydrogen peroxide molecules (cf. Scheme 1), or due to the decomposition of H2O2 by Cu(I) complex through Fenton type reaction, the Weissberger system became one of the most potent generators of hydroxyl radicals. Scheme 1. Generation of H2O2 via the Weissberger system composed of ascorbate and Cu(II) under aerobic conditions. AscH − represents ascorbate anion and DHA represents dehydroascorbate. When the Weissberger system has no additional oxidizable substrate, we speak about ascorbate autoxidation. In any case, it should be pointed out that the biological consequences of interactions of vitamin C with biogenic transition metal cations of iron, copper, or manganese have not been fully understood yet. Over the past decade, the prooxidant properties of ascorbate have been investigated in addition to its better explored antioxidant role. When oxidizable substrates are simultaneously present, the reaction products consist of both oxidized/decomposed substrateand DHA-dehydroascorbate, whose molecules in aqueous milieus hydrolyze fast to 2,3-diketo-L-gulonic acid. Although for the next sections of this minireview the complexation of ascorbate with another biogenic transition metal is not essential, the reader could find some complexation kinetics and/or equilibrium data between ascorbate and several metal cations, e.g., in the paper by Fornaro and Coichev. One of the frequently cited statements in literature is that copper cations released from the ceruloplasmin macromolecule are extensively entrapped by albumin present in the bloodstream. This tenet is naturally in part true, but it must also be admitted that the anions of ascorbate are the most probably co-responsible for the release of copper cations from ceruloplasmin. The charge-charge interaction between AscH − and, e.g., [CuCl] + with a rate constant of 280 mol −1 L −1 s −1may outweigh the rate of gradual dissociation of the complex between [AscH] − and [CuCl] + followed by a subsequent association of Cu(II) with a copper fixing/binding site on the albumin molecule.) Scheme 1. Generation of H 2 O 2 via the Weissberger system composed of ascorbate and Cu(II) under aerobic conditions. AscH − represents ascorbate anion and DHA represents dehydroascorbate. When the Weissberger system has no additional oxidizable substrate, we speak about ascorbate autoxidation. In any case, it should be pointed out that the biological consequences of interactions of vitamin C with biogenic transition metal cations of iron, copper, or manganese have not been fully understood yet. Over the past decade, the pro-oxidant properties of ascorbate have been investigated in addition to its better explored antioxidant role. When oxidizable substrates are simultaneously present, the reaction products consist of both oxidized/decomposed substrateand DHA-dehydroascorbate, whose molecules in aqueous milieus hydrolyze fast to 2,3-diketo-L-gulonic acid. Although for the next sections of this minireview the complexation of ascorbate with another biogenic transition metal is not essential, the reader could find some complexation kinetics and/or equilibrium data between ascorbate and several metal cations, e.g., in the paper by Fornaro and Coichev. One of the frequently cited statements in literature is that copper cations released from the ceruloplasmin macromolecule are extensively entrapped by albumin present in the bloodstream. This tenet is naturally in part true, but it must also be admitted that the anions of ascorbate are the most probably co-responsible for the release of copper cations from ceruloplasmin. The charge-charge interaction between AscH − and, e.g., [CuCl] + with a rate constant of 280 mol −1 L −1 s −1may outweigh the rate of gradual dissociation of the complex between [AscH] − and [CuCl] + followed by a subsequent association of Cu(II) with a copper fixing/binding site on the albumin molecule.) ## Hyaluronan-oxidizable biological substrate Hyaluronic acid (hyaluronan; HA;, or its salts, is a linear high-molar-mass natural polysaccharide formed from disaccharide units of regularly alternating N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA) units linked by β-(1→3) and β-(1→4) linkages. The chemical structure of HA is regular, the only exception is a possible replacement of N-acetyl-D-glucosamine by deacetylated glucosamine residues. Pharmaceutics 2021, 13, 1815 3 of 13 ## Hyaluronan-oxidizable biological substrate Hyaluronic acid (hyaluronan; HA;, or its salts, is a linear high-molar-mass natural polysaccharide formed from disaccharide units of regularly alternating N-acetyl-D-glucosamine (GlcNAc) and D-glucuronic acid (GlcA) units linked by β-(1→3) and β-(1→4) linkages. The chemical structure of HA is regular, the only exception is a possible replacement of N-acetyl-D-glucosamine by deacetylated glucosamine residues. A human body weighing about 70 kg contains approx. 15 g of HA, whereas one third of this amount is turned over every day. Such an unusually extensive de novo synthesis of HA megadalton macromolecules suggests that the functions of both native hyaluronan and its lower sized fragments in the body will be diverse: In the body of vertebrates HA is abundantly present in almost all body fluids and tissues. In a fibrous tissue a capsule called synovium, one of the main components is the synovial fluid (SF), functions as a lubricant. The SF in a healthy human being, along with the blood plasma filtrate, also contains the entangled macromolecules of HA {1.4-3.6 mg/mL}. While in SF and vitreous humor HA macromolecules are not associated with proteins, the HA chains, filling the space between the collagen fibrils, provide elastic properties of these soft tissues. Yet, HA in extracellular matrices is linked with several proteoglycans creating a scaffold in the forms of more or less hard tissues such as skin, umbilical cord, and cartilage. Two ways of HA decay must be denoted here: first, enzymatic depolymerization, by which HA fragments of lower molar mass are formed, and second, oxidative degradation, a process which is of great interest to polymer chemists, biochemists and molecular biologists. The half-life of HA depolymerization (by hyaluronidases) is in the range of 1-3 weeks in cartilage, 1-2 days in skin and only 2-5 min in blood plasma. In contrast, in SF of healthy individuals, whose fluid lacks any hyaluronidases, the half-life of HA ≈ 12 h is suggestive of other than enzymatic decay. Such a rapid turnover of HA in SF could be elucidated by a mechanical pumping-out of a part of SF through the lymphatic system during the day-time moving activity of a person. Another proposed mechanism is an oxidative degradation of several megadalton-HAs in SF of healthy individuals. This tenet is supported indirectly by the fact that under physiological conditions, the concentration of ascorbate in SF of healthy humans reaches the values closely to those in blood plasma, i.e., 40-140 µmol/L.illustrates the action of oxidative system comprising complex of ascorbate---Cu(II/Cu(I)---dioxygen: As evident, a megadalton-HA sample progressively degrades to intermediate-sized polymer fragments, which mean molar mass is reduced by one or even two orders of magnitude. -[-GlcNAc-GlcA-GlcNAc-GlcA-]n - A human body weighing about 70 kg contains approx. 15 g of HA, whereas one third of this amount is turned over every day. Such an unusually extensive de novo synthesis of HA megadalton macromolecules suggests that the functions of both native hyaluronan and its lower sized fragments in the body will be diverse: In the body of vertebrates HA is abundantly present in almost all body fluids and tissues. In a fibrous tissue a capsule called synovium, one of the main components is the synovial fluid (SF), functions as a lubricant. The SF in a healthy human being, along with the blood plasma filtrate, also contains the entangled macromolecules of HA {1.4-3.6 mg/mL}. While in SF and vitreous humor HA macromolecules are not associated with proteins, the HA chains, filling the space between the collagen fibrils, provide elastic properties of these soft tissues. Yet, HA in extracellular matrices is linked with several proteoglycans creating a scaffold in the forms of more or less hard tissues such as skin, umbilical cord, and cartilage. Two ways of HA decay must be denoted here: first, enzymatic depolymerization, by which HA fragments of lower molar mass are formed, and second, oxidative degradation, a process which is of great interest to polymer chemists, biochemists and molecular biologists. The half-life of HA depolymerization (by hyaluronidases) is in the range of 1-3 weeks in cartilage, 1-2 days in skin and only 2-5 min in blood plasma. In contrast, in SF of healthy individuals, whose fluid lacks any hyaluronidases, the half-life of HA ≈ 12 h is suggestive of other than enzymatic decay. Such a rapid turnover of HA in SF could be elucidated by a mechanical pumping-out of a part of SF through the lymphatic system during the day-time moving activity of a person. Another proposed mechanism is an oxidative degradation of several megadalton-HAs in SF of healthy individuals. This tenet is supported indirectly by the fact that under physiological conditions, the concentration of ascorbate in SF of healthy humans reaches the values closely to those in blood plasma, i.e., 40-140 µmol/L.illustrates the action of oxidative system comprising complex of ascorbate-Cu(II/Cu(I)-dioxygen: As evident, a megadalton-HA sample progressively degrades to intermediate-sized polymer fragments, which mean molar mass is reduced by one or even two orders of magnitude. ## Hyaluronan oxidative degradation by free-radical-chain reaction ## Initiation reaction(s) As shown in the sub-section "Sequestration of copper cations with ascorbate" for the generation of - OH radical(s) the so called Weissberger biogenic oxidative system can produce a continual flux of hydroxyl radicals. (To simplify some reaction sequences appearing in this minireview, HA abbreviates the hyaluronan macromolecule. The A - denotes a C-centered hyaluronan macroradical, a peroxy-type macroradical AOO - along with a highly unstable alkoxy-type macroradical AO - represents O-centered intermediate macroradicals). ## Hyaluronan oxidative degradation by free-radical-chain reaction ## Initiation reaction(s) As shown in the sub-section "Sequestration of copper cations with ascorbate" for t generation of - OH radical(s) the so called Weissberger biogenic oxidative system can p duce a continual flux of hydroxyl radicals. (To simplify some reaction sequen appearing in this minireview, HA abbreviates the hyaluronan macromolecule. The A - d notes a C-centered hyaluronan macroradical, a peroxy-type macroradical AOO - alo with a highly unstable alkoxy-type macroradical AO - represents O-centered intermedi macroradicals.) The following Scheme 2 describes structural chemical formulae the phase of initiati reaction(s) of high-molar-mass HA oxidative degradation: Scheme 2. An intact HA macromolecule (upper panel) reacts with - OH radical yielding an interm diate A - , i.e., a C-centered hyaluronan macroradical (lower panel), which immediately reacts w ## Hyaluronan oxidative degradation by free-radical-chain reaction ## Initiation reaction(s) As shown in the sub-section "Sequestration of copper cations with ascorbate" for the generation of - OH radical(s) the so called Weissberger biogenic oxidative system can produce a continual flux of hydroxyl radicals. (To simplify some reaction sequences appearing in this minireview, HA abbreviates the hyaluronan macromolecule. The A - denotes a C-centered hyaluronan macroradical, a peroxy-type macroradical AOO - along with a highly unstable alkoxy-type macroradical AO - represents O-centered intermediate macroradicals.) The following Scheme 2 describes structural chemical formulae the phase of initiation reaction(s) of high-molar-mass HA oxidative degradation: Scheme 2. An intact HA macromolecule (upper panel) reacts with - OH radical yielding an intermediate A - , i.e., a C-centered hyaluronan macroradical (lower panel), which immediately reacts with a molecule of oxygen resulting in a peroxy-type macroradical AOO - . Scheme 2. An intact HA macromolecule (upper panel) reacts with - OH radical yielding an intermediate A - , i.e., a C-centered hyaluronan macroradical (lower panel), which immediately reacts with a molecule of oxygen resulting in a peroxy-type macroradical AOO - . The macroradical AOO - can undergo the reaction with Cu(I) complex (cf. Scheme 1) yielding a highly unstable alkoxy-type macroradical. It is comprehensible that either AOO - macroradical or that of AO - , along with the parent A - intermediate macroradical, form a collection of highly reactive compounds, i.e., the initiators of subsequent self-perpetuating free-radical degradation of high-molar-mass HA. ## Transfer of the free-radical centre and fragmentation reaction(s) As is well-known in macromolecular chemistry, a long chain alkoxy-type macroradical freely undergoes the strand scission due to the β-cleavage. Thus the scission at, e.g., C(1) on Pharmaceutics 2021, 13, 1815 5 of 13 the ring of D-glucuronic acid, yields polymer fragments, namely a macromolecule bearing a terminal C=O group and a novel alkoxy-type macroradical (cf. Scheme 3). As a rule of such a degradation reaction both fragments have reduced molar mass. form a collection of highly reactive compounds, i.e., the initiators of subsequent selfpetuating free-radical degradation of high-molar-mass HA. ## Transfer of the free-radical centre and fragmentation reaction(s) As is well-known in macromolecular chemistry, a long chain alkoxy-type macro ical freely undergoes the strand scission due to the β-cleavage. Thus the scission at, C(1) on the ring of D-glucuronic acid, yields polymer fragments, namely a macromole bearing a terminal C=O group and a novel alkoxy-type macroradical (cf. Scheme 3). A rule of such a degradation reaction both fragments have reduced molar mass. Scheme 3. The long chain alkoxy-type macroradical AO - undergoes a transfer of one electro e.g., C(1) on the ring of D-glucuronic acid (upper panel). The decay of the native chain alkoxymacroradical AO - yields two polymer fragments (lower panel) having reduced molar masses. newly formed alkoxy-type macroradical of 38shorter molar size (AO - ) naturally could act a initiator of the self-perpetuating free-radical HA degradation. The initiating - OH radical(s) (cf. Scheme 1) can react with the D-glucuronate/Dcuronic acid and N-acetyl-D-glucosamine functional moieties by opening the alkyl rwithout breaking the HA chain. ## Termination reaction(s) The free-radical degradation reactions of the native high-molar-mass hyaluronan long to self-perpetuating reactions. One of the most effective procedures terminating self-perpetuating reactions is to scavenge this sequence in the very phase of their in tion. Reactions represented in Scheme 1, subsequently followed by H2O2 decomposit can be terminated by a so-called preventive antioxidant, e.g., a substance freely dona an atom of hydrogen, i.e., - H: In such a case the substance, classifiable as HAT-hydro atom transferring, terminates the action of the - OH radical and thus it interrupts the eration of A - macroradical. (It should be claimed here that at pH 7.4, more than 99.9% ascorbic acid (AscH2) is present dissociated as AscH − . Thus, the antioxidant chemistr vitamin C is the chemistry of ascorbate, which freely donates a hydrogen atom to an dizing radical.) According to the cascade of reactions showed in Schemes 2 and 3 it is obvious, to prevent the propagation phase of the free-radical HA degradation one must appl antioxidant which acts as HAT. Such a property is attributed to the so-called chain-br ing antioxidants. It is comprehensible that the antioxidant acting as chain-breaking sho effectively scavenge both the AOO - and AO - radicals. In case of these two O-cente . ## Scheme 3. The long chain alkoxy-type macroradical AO - undergoes a transfer of one electron at, e.g., C(1) on the ring of D-glucuronic acid (upper panel). The decay of the native chain alkoxy-type macroradical AO - yields two polymer fragments (lower panel) having reduced molar masses. The newly formed alkoxy-type macroradical of shorter molar size (AO - ) naturally could act as the initiator of the self-perpetuating free-radical HA degradation. The initiating - OH radical(s) (cf. Scheme 1) can react with the D-glucuronate/Dglucuronic acid and N-acetyl-D-glucosamine functional moieties by opening the alkyl ringswithout breaking the HA chain. ## Termination reaction(s) The free-radical degradation reactions of the native high-molar-mass hyaluronan belong to self-perpetuating reactions. One of the most effective procedures terminating the self-perpetuating reactions is to scavenge this sequence in the very phase of their initiation. Reactions represented in Scheme 1, subsequently followed by H 2 O 2 decomposition, can be terminated by a so-called preventive antioxidant, e.g., a substance freely donating an atom of hydrogen, i.e., - H: In such a case the substance, classifiable as HAT-hydrogen atom transferring, terminates the action of the - OH radical and thus it interrupts the generation of A - macroradical. (It should be claimed here that at pH 7.4, more than 99.9% of ascorbic acid (AscH 2 ) is present dissociated as AscH − . Thus, the antioxidant chemistry of vitamin C is the chemistry of ascorbate, which freely donates a hydrogen atom to an oxidizing radical). According to the cascade of reactions showed in Schemes 2 and 3 it is obvious, that to prevent the propagation phase of the free-radical HA degradation one must apply an antioxidant which acts as HAT. Such a property is attributed to the so-called chain-breaking antioxidants. It is comprehensible that the antioxidant acting as chain-breaking should effectively scavenge both the AOO - and AO - radicals. In case of these two O-centered radicals, although their standard reduction potential is lower than that for hydroxyl radical - OH, H + /H 2 O (+2.31 V), they are still very oxidative-similar to those of an aliphatic peroxyl radical ROO - , H + /ROOH (≈ +1.0 V) or an aliphatic alkoxyl radical RO - , H + /ROH (≈ +1.6 V). ## Assessment the substance antioxidative profile by hyaluronan plus the weissberger system The idea to use high-molar-mass HA for the evaluation of antioxidants can be traced to the year 1994. At present, the experimental set-up uses the physiologic solution of the HA sample with an average molar mass ≈ 1.5 MDa. The time-dependent changes in dynamic-viscosity values after the application of CuCl 2 and ascorbic acid are monitored by a rotation viscometry device. In the experimental design when the application of the test substance precedes the addition of ascorbic acid, one examines the preventive antioxidant properties of the substance. On assessing the substance chain-breaking antioxidant properties the addition of substance examined follows the application of ascorbic acid after a properly selected time interval, i.e., during the steady state phase of HA degradation propagation. The panels inschematically illustrate the types of functional time dependencies on dynamic viscosity (η). (Other methods used were infrared spectroscopy, thermal chemiluminescenceand EPR spectroscopy, the last one to identify the formation of radicals in selected oxidation systems.) To comment the below represented experimental results it should be claimed that, as repeatedly proved, the decrease in dynamic viscosity values reflects the decrease in average molar mass of the HA sample with respect to the existing functional dependence η = f (M). ## Assessment the substance antioxidative profile by hyaluronan plus the weissberger system The idea to use high-molar-mass HA for the evaluation of antioxidants can be traced to the year 1994. At present, the experimental set-up uses the physiologic solution of the HA sample with an average molar mass ≈ 1.5 MDa. The time-dependent changes in dynamic-viscosity values after the application of CuCl2 and ascorbic acid are monitored by a rotation viscometry device. In the experimental design when the application of the test substance precedes the addition of ascorbic acid, one examines the preventive antioxidant properties of the substance. On assessing the substance chain-breaking antioxidant properties the addition of substance examined follows the application of ascorbic acid after a properly selected time interval, i.e., during the steady state phase of HA degradation propagation. The panels inschematically illustrate the types of functional time dependencies on dynamic viscosity (η). (Other methods used were infrared spectroscopy, thermal chemiluminescenceand EPR spectroscopy, the last one to identify the formation of radicals in selected oxidation systems.) To comment the below represented experimental results it should be claimed that, as repeatedly proved, the decrease in dynamic viscosity values reflects the decrease in average molar mass of the HA sample with respect to the existing functional dependence η = f (M). The time dependence of the decrease in the value of dynamic viscosity marked in black on panels A to E of the experimental set-up is as follows: CuCl2 and ascorbic acid solution are gradually applied to the HA solution so that the actual concentrations of these components are 2 mg/mL, 1 and 100 µmol/L, respectively. It should also be noted here that the experiments were performed under aerobic conditions, i.e., the concentration of oxygen in aqueous solutions, at standard barometric pressure has been ≈ 250 µmol/L at 25 °C. When verifying whether a given substance acts as a preventive antioxidant, it was applied in several doses, so that the actual concentration of the substance within the experimental vessel was usually 100 (red), 10 (blue), and 1 (green curve) µmol/L, respectively. The results of antioxidative profile assessment of several substances are given in. The time dependence of the decrease in the value of dynamic viscosity marked in black on panels A to E of the experimental set-up is as follows: CuCl 2 and ascorbic acid solution are gradually applied to the HA solution so that the actual concentrations of these components are 2 mg/mL, 1 and 100 µmol/L, respectively. It should also be noted here that the experiments were performed under aerobic conditions, i.e., the concentration of oxygen in aqueous solutions, at standard barometric pressure has been ≈250 µmol/L at 25 - C. When verifying whether a given substance acts as a preventive antioxidant, it was applied in several doses, so that the actual concentration of the substance within the experimental vessel was usually 100 (red), 10 (blue), and 1 (green curve) µmol/L, respectively. The results of antioxidative profile assessment of several substances are given in.a N-Acetylcysteine, L-cysteine, and L-glutathione are weak acids, which pK a values of their −SH group equal to 9.52, 10.78, and 9.65, respectively. Due to dissociation of thiol a minute fraction of thiolate anions along with the generated thiyl radicals act both like the reductants of O 2 molecules yielding superoxide anion radicals, which subsequently participate in reaction with in situ generated H 2 O 2 molecules according to the reversible Haber-Weiss reaction O 2 - − + H 2 O 2 ↔HO - + HO − + O 2. b Since both substances, i.e., cemtirestat and isatin strongly intercalate cupric ions, no redox active Cu(II) was available to form ascorbate-Cu(II)/Cu(I)-dioxygen complex even at the lowest substance concentration (1/100). c Mn(III)meso-tetrakis(N-ethylpyridinium-2-yl)porphyrin (MnTE-2-PyP 5+ ), in phase II clinical trials, accepts one electron from AscH − and subsequently the Mn(II) center acts as a reductant of O 2 molecules yielding superoxide anion radical, which subsequently participate in reaction with in situ generated H 2 O 2 molecules according to the above mentioned reversible Haber-Weiss reaction. ## Concluding remarks We have reported evidence to support our thesis regarding the assessment of the antioxidative efficiency of an endo-or exogenous water soluble substance by exploiting a reaction system comprising high-molar-mass hyaluronan along with cupric cations and ascorbate. The ascorbate level in body fluids of healthy individuals is in the range of 40-140 µmol/L. The total concentrations of copper ions in healthy human beings may reach micromolar levels. Thus during ascorbate (auto)-oxidation in the presence of trace levels of cupric ions as catalysts, direct transformation of O 2 to H 2 O 2 happens. Subsequently, the produced hydrogen peroxide is decomposed by the action of the transition metal counter cuprous cations, which are site-specifically fixed by the HA polyanionic chain and thus - OH radicals interact with HA macromolecules. The generated hydroxyl radicals, and/or mainly the C-centered hyaluronan macroradicals, if not scavenged by a proper preventive antioxidant, continue their subsequent self-perpetuating free-radical degradation up to the moment of their reaction with a chainbreaking antioxidant. # Data availability statement: The data presented in this study is openly available.

One-year results of a clinical trial of olipudase alfa enzyme replacement therapy in pediatric patients with acid sphingomyelinase deficiency ## Clinical description and management of patient with anaphylactic reaction Summary A 17-month-old patient included in the Infant/early child cohort experienced a serious adverse event of urticaria considered an anaphylactic reaction that necessitated stopping treatment until a desensitization protocol was implemented. The patient was heterozygous for two variants previously identified as likely pathogenic, neither of which was the common p.Arg610del variant. Clinical history included allergy or potential allergic reactions for eczema. After undergoing the desensitization protocol, the patient was able to reach the target dose of 3 mg/kg and continues receiving olipudase alfa infusions in the long-term extension study. Intermittent events of vomiting, pyrexia and urticaria have occurred at subsequent post-desensitization infusions, and the patient receives prophylactic treatment with oral diphenhydramine hydrochloride, IV diphenhydramine hydrochloride, and/or IV methylprednisolone at each olipudase alfa infusion. The patient had an intermediate IgG anti-drug antibody (ADA) response and was positive for IgE ADA but did not develop neutralizing antibodies that inhibited either enzyme activity or cellular uptake. ## Clinical details The patient had events of mild vomiting considered infusion associated reactions (IARs) at the Week 4 visit after receiving 0.3 mg/kg olipudase infusion, at the Week 8 visit after receiving 0.6 mg/kg, and at the Week 10 visit after receiving the second 0.6 mg/kg dose. Vomiting was managed with ondansetron administration. Mild events of pyrexia also occurred post-infusion after the Week 8 and Week 10 visits (body temperature of 38.3°C and 38°C, respectively) and were treated with oral acetaminophen and/or ibuprofen. At the Week 10 visit, the patient also had an adverse event of urticaria considered an IAR (diffused on face, torso, and extremities) of mild intensity 42 minutes after the start of infusion, during infusion at a rate of 10 mL/hr. Olipudase alfa was temporarily interrupted, and intravenous (IV) diphenhydramine hydrochloride 11 mg (1 mg/kg) administered. The urticaria resolved within 68 minutes, and the olipudase alfa infusion was restarted and completed at a decreased rate of 5 mL/hr. Pre infusion vital signs showed heart rate at 129 beats per minute (bpm), blood pressure (BP) at 116/74 mmHg, and body temperature at 37.0°C. At 1-hour post infusion, heart rate was 121 bpm, BP as 132/86, and body temperature was 38.0°C. Following the event of urticaria, the Investigator decided to repeat the dose of 0.6 mg/kg at the next infusion at Week 12. At the Week 12 visit, during the infusion of 0.6 mg/kg olipudase alfa, the patient experienced an anaphylactic reaction of severe intensity 27 minutes after beginning infusion during the infusion rate of 10 mL/hr. Heart rate and blood pressure at the time of the event were 186 bpm and 131/71, respectively. The infusion was stopped and corrective treatment administered including intramuscular (IM) epinephrine 0.11 mg, IV methylprednisolone sodium succinate 10.8 mg, and IV diphenhydramine hydrochloride 11 mg. This serious adverse event was assessed as related to olipudase alfa by the Investigator and considered an IAR. ## Immunological assessment Anti-olipudase alfa seroconversion was observed for this patient at Week 10 (IgG ADA titer=400) and the peak IgG ADA titer during the study was 1600 at Week 12 (corresponding to an intermediate ADA response). At the end of the study (Week 64), the IgG ADA titer was 100. The patient did not develop during the study neutralizing antibodies that inhibited either enzyme activity or cellular uptake. Additional immunological testing, performed at the Week 12 visit because of the event of anaphylactic reaction of severe intensity, showed positive anti olipudase alfa IgE antibodies preinfusion (at 1.30 IU/mL; normal reference range <0.35 IU/mL), and post-infusion serum tryptase of 6.7 µg/L (normal reference range ≤12.5 µg/L). Circulating immune complex was 0.8 µg eq/mL post-infusion and complement activation was negative. ## Desensitization protocol and outcomes The patient did not receive any olipudase alfa infusions from Week 14 through Week 26 included to allow for preparation and implementation of a desensitization procedure for restarting olipudase alfa infusions. Olipudase alfa was restarted at a dose of 0.3 mg/kg at Week 28 at a 1:100,000 dilution of the concentration at the time of the anaphylactic reaction, with the addition of a coating solution to prevent adherence of olipudase alfa to the tubing at dilute concentrations. After the first restarting dose, escalation of concentrations proceeded slowly and gradually and was adjusted based on patient tolerance to infusions, which was carefully monitored. As a precautionary measure, Week 28 and Week 30 infusions were done at the pediatric intensive care unit. The patient reached the target maintenance dose of 3 mg/kg at Week 50 and the standard infusion administration procedure was progressively reinstated and fully followed at Week 58. participant received oral diphenhydramine hydrochloride, IV diphenhydramine hydrochloride, and/or IV methylprednisolone as prophylactic treatments at each olipudase alfa infusion. During the Week 46 visit infusion of a 2.0 mg/kg dose, the patient had an adverse event of urticaria of mild intensity (scattered in the right diaper area, on torso, underarms, calves, and shins) four hours after the start of infusion. Infusion was temporarily interrupted and IV diphenhydramine hydrochloride 13 mg (1mg/kg) was administered. The event of urticaria resolved and the infusion was restarted and completed at the same rate of 20 mL/hr. # Conclusion Patients showing signs of severe hypersensitivity reactions following olipudase alfa infusions can be successfully managed with a desensitization protocol. Although not routinely recommended, in this case the investigator elected to use pre-treatment prophylaxis with corticosteroids and H1-receptor antagonists to minimize IARs.

ZODET: Software for the Identification, Analysis and Visualisation of Outlier Genes in Microarray Expression Data Complex human diseases can show significant heterogeneity between patients with the same phenotypic disorder. An outlier detection strategy was developed to identify variants at the level of gene transcription that are of potential biological and phenotypic importance. Here we describe a graphical software package (z-score outlier detection (ZODET)) that enables identification and visualisation of gross abnormalities in gene expression (outliers) in individuals, using whole genome microarray data. Mean and standard deviation of expression in a healthy control cohort is used to detect both over and under-expressed probes in individual test subjects. We compared the potential of ZODET to detect outlier genes in gene expression datasets with a previously described statistical method, gene tissue index (GTI), using a simulated expression dataset and a publicly available monocyte-derived macrophage microarray dataset. Taken together, these results support ZODET as a novel approach to identify outlier genes of potential pathogenic relevance in complex human diseases. The algorithm is implemented using R packages and Java.Availability: The software is freely available from # Introduction Many human diseases, such as inflammatory bowel disease and type 1 diabetes, are complex, multifactorial syndromes with genetic and environmental determinants. Substantial heterogeneity with regards to causation and disease progression exists between individual patients with the same phenotypic disorder. It has been postulated that rare or low frequency variants, structural rearrangements such as deletions, insertions, translocations, and epigenetic variation could be important in the pathogenesis of these complex disorders and account for the observed heterogeneity [bib_ref] Finding the missing heritability of complex diseases, Manolio [/bib_ref]. All of these are incompletely assessed by current genome wide association studies (GWAS). Many of these genetic changes would be expected to be associated with alterations in gene expression, possibly of large biological effect, ultimately giving rise to phenotypic abnormalities. A recent paper combined population-scale human genomic sequence data with transcriptomic data and identified an enrichment of rare variants associated with outlier gene expression [bib_ref] Rare and common regulatory variation in population-scale sequenced human genomes, Montgomery [/bib_ref]. They concluded that across multiple tissues and developmental stages, an individual would be expected to have hundreds of rare variants with large effects on gene expression. The examination of significantly overexpressed genes in individual patients (or subgroups of patients) has successfully been employed in the field of cancer genomics [bib_ref] Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer, Tomlins [/bib_ref]. There are a number of methods for outlier detection currently in the literature, such as the gene tissue index (GTI), cancer outlier profile analysis (COPA) and outlier robust test (ORT), each of which use different algorithms in order to identify probes that are abnormally expressed in subgroups of patients [bib_ref] GTI: a novel algorithm for identifying outlier gene expression profiles from integrated..., Mpindi [/bib_ref] [bib_ref] Cancer outlier differential gene expression detection, Wu [/bib_ref] [bib_ref] COPA-cancer outlier profile analysis, Macdonald [/bib_ref]. Here we describe a software package based on z-score outlier detection (ZODET) that enables identification of potentially biologically relevant abnormalities in gene expression (outliers) in individuals with complex disorders compared with a comparison population, using whole genome microarray data. By concentrating on individual outliers, we provide a valuable addition to commonly used microarray analysis tools, such as SAM [bib_ref] Significance analysis of microarrays applied to the ionizing radiation response, Tusher [/bib_ref]. # Materials and methods ## Software implementation Implementation of this software has used two programming technologies: The R statistical programming environment (http:// www.R-project.org), utilising the Biobase package from the Bioconductor platform; and the Java programming language [bib_ref] Bioconductor: open software development for computational biology and bioinformatics, Gentleman [/bib_ref]. The analysis can be run via a configurable Graphical User Interface (GUI) or on the command line . Installation and configuration instructions are provided in the technical documentation supplied with the software (http://www.ucl.ac.uk/ medicine/molecular-medicine/publications/microarray-outlier-analysis). The software is freely available and can be run on either Windows or Mac OSX operating systems. The purpose of this software is the identification and visual analysis of outlier probes (or genes) from microarray gene expression data. To identify the potential outliers a control group of samples and the experimental samples are defined prior to analysis . Currently, the software supports five alternative methods for defining which sub-set of individual samples the potential outliers may occur in and the group of samples (control group) which each is compared to. These five methods of comparison are: For each of the samples selected for outlier analysis, an iterative procedure is carried out on the expression data. Two adjustable thresholds (which both have to be met) are used to identify the probe outliers: (i) the significance level of the standardised deviation of the expression levels from the experimental sample, when compared to the average (mean) expression levels of the control group; and (ii) the (log 2 ) fold-change between the expression level from the experimental sample and the average of the control group. This method assumes that the expression values are normally distributed. Therefore, caution should be taken to ensure the normality of the data, especially when using small sample sizes (e.g., less than 30). . The Graphical User Interface (GUI) allows the user to set the analysis parameters, the required fold change, statistical test (p-value, q-value or Bonferroni corrected p-value) and statistical threshold. The analysis can be conducted using the whole experimental group or a specific individual can be chosen. doi:10.1371/journal.pone.0081123.g001 To assess the significance of the standardised deviation of the experimental sample expression values from the control group average, a Z-score is calculated for each probe (or feature) on the microarray: [formula] Z~E sample {m sð1Þ [/formula] Where: E sample is the expression level of the microarray gene probe for the experimental sample; and m and s are the mean and standard deviation of the expression levels of the gene probe for samples in the control group. From the Z-scores, p-values are then calculated with reference to a standard normal distribution where the p-value is the probability that the probe z-score of the sample being tested falls within the distribution of the control group samples. These p-values -or those corrected for multiple hypothesis testing, on a per gene basis, using either the Bonferroni or q-value method -can be used to assess the significance [bib_ref] Statistical methods for identifying differentially expressed genes in DNA microarrays, Storey [/bib_ref]. The default p-value threshold is 0.01, but this can be easily modified before each analysis run. The fold-change between the expression level of the experimental sample, and the control group, is calculated for each probe on the array using the following method. [formula] foldC~E sample {mð2Þ [/formula] This provides an additional measure of the difference in the expression level of the experimental and the control groups and also determines whether the potential outlier-probe is under or . Overview of experimental design and output from the ZODET analysis. A. Microarray experimental design will contain two groups, a control group which will provide the data for the normal distribution of each probe and an experimental group (C1-3). The software runs each test group member independently against the control group and identify probes that are expressed at levels significantly outside the normal distribution. B. For each experimental subject the software generates a scatter plot of all of the expressed probes from subject C1 against the mean value from the control group. Probes that are classified as up-or down-regulated are highlighted in red and green, respectively. A Volcano plot is generated to visualise p-value and fold change information for all probes, with over and under expressed outliers highlighted in red and green, respectively. The vertical and horizontal lines represent the fold-change and p-value thresholds used, respectively. A combined dendrogram and heatmap shows the expression level of probes identified as down-regulated in C1, compared to the control group and two additional experimental subjects (C2 and C3). C. The software also generates outlier analysis results for all experimental subjects (C1-3). The total up-regulated and downregulated probes are tabulated for all test subjects along with the results for the three available statistical tests. Combined dendrogram and heatmaps are generated for all of the up-regulated and down-regulated probes identified and hierarchical clustering on both probes and subjects performed. Over and under expressed probes are highlighted in red and green, respectively. doi:10.1371/journal.pone.0081123.g002 over expressed, relative to the control group. In equation 2: foldC is the fold-change; E sample is the expression level of the test sample; and m is the mean expression level of the control group. To correctly estimate the fold-change between sample classes all gene expression levels used as input to this software should be in normalised log 2 format. The software is designed to not be microarray platform specific and can be used to analyse any log 2 transformed microarray expression data (e.g., from Affymetrix, Illumina or other arrays). The default fold-change threshold is 1, which can be easily modified before each analysis run. User defined annotation files can also be used to associate the microarray probes with the relevant gene descriptions. To aid the interpretation and visualisation of the results a number of clearly formatted graphs and tables are automatically generated by the software during the analysis, including: For each individual ## Patient recruitment These studies were approved by the Joint UCL/UCLH Committee for the Ethics of Human Research (project numbers 02/0324). Forty volunteers were recruited from University College London Hospital (UCLH)/University College London (UCL). Written consent was obtained from all volunteers. ## Monocyte derived macrophage isolation and culture Peripheral venous blood was collected from subjects into syringes containing 5 U/ml heparin. Mononuclear cells were isolated by differential centrifugation (800 g, 30 min, 20uC) over Lymphoprep and washed twice with sterile phosphate-buffered saline (PBS; GIBCO, Paisley, UK) at 500 g (5 min, 20uC). Cells were resuspended in 10 ml RPMI-1640 medium (Invitrogen) supplemented with 100 U/ml of penicillin (GIBCO), 100 mg/ml streptomycin (GIBCO) and 20 mM HEPES pH 7.4 (Sigma-Aldrich), and plated at a density of approximately 5610 6 cells/ml in 8 cm 2 Nunclon TM Surface tissue culture dishes (Nunc, Roskilde, Denmark) at 37uC, 5% CO 2 . After 2 h, non-adherent cells were discarded and 10 ml of fresh RPMI supplemented with 10% foetal bovine serum (FBS; Sigma) added to each tissue culture dish. Cells were then cultured for 5 days at 37uC, 5% CO2, with the addition of a further 10 ml of fresh 10% FBS/RPMI after 24 h. Cells were then washed twice in PBS, scraped and spun down at 500 g (5 min, 20uC). Cells were resuspended into X-vivo-15 medium (Cambrex, MD, USA) and plated at a density of 10 6 cells per 8 cm 2 Nunclon TM dish for a further 25 h at 37uC, 5% CO2. ## Rna purification Total RNA was prepared from monocyte-derived macrophages, using the RNeasy Mini Kit with RNase-free DNase treatment (Qiagen GmbH, Hilden, Germany). Optical density readings were determined for OD 260 /OD 280 and OD 260 /OD 230 using a NanoDrop ND-1000 spectrophotometer (Fisher Scientific, Loughborough, UK) to assess protein and solvent contamination respectively. ## Whole genome microarray analysis For each sample, 500 ng of total RNA was amplified and purified using the Illumina TotalPrep-96 RNA Amplification kit (Ambion, UK), according to the manufacturer's instructions. Biotin-Labelled cRNA was then normalised to a concentration of 150 ng/ml and 750 ng was hybridised to Illumina Human-WG6 v3.0 Expression BeadChips (Illumina CA, USA) for 16 h at 58uC. Following hybridisation, beadarrays were washed and stained with streptavidin-Cy3 (GE Healthcare, UK). Beadarrays were scanned using the Beadarray reader and image data was then processed using Genome Studio software (Illumina, CA, USA). The cubic spline normalised data and subject information can be found at http://www.ucl.ac.uk/medicine/molecular-medicine/publications/ microarray-outlier-analysis. The microarray data has also been deposited in the Gene Expression Omnibus (GEO) under accession GSE51256. Due to the relatively low sample sizes, the dataset was tested for normality using the lillie.test function from the R-package nortest (http://cran.r-projects.org/web/packages/nortest/) [bib_ref] An Analytic Approximation to the Distribution of Lilliefors's Test Statistic for Normality, Dallal [/bib_ref]. All 20,019 probes on the array were assessed for normality using a p-value of 0.05, followed by correction for multiple testing. This resulted in a very small number of probes (240) found to be not normally distributed indicating that the vast majority of probes are normally distributed and can therefore be used as input to the ZODET method. ## Genomic dna extraction, pcr verification and sequencing Peripheral blood samples were collected in an ethylenediaminetetraacetic acid disodium salt vacutainer (BD Bioscience, UK) and gDNA extracted using the QIAamp DNA blood Mini Kit (Qiagen GmbH), in accordance with the manufacturer's instructions. PCR verification of the chromosome translocation involving G-protein receptor-128 (GPR128) and TRK-fused gene (TFG) was determined using TFG forward primer CCACAGCCTACCTGT-GAGTG, GPR128 reverse primer TGGGTTGTTTGTGGAAAT. Verification of the intact GPR128 gene was performed using the reverse primer listed for the translocation in combination with forward primer GCAGGCTTTCTTTCTTGAGG. A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (rs10774671) within 29-59-oligoadenylate synthetase 1 (OAS1) was determined following the method describes previously [bib_ref] Variation in antiviral 29,59-oligoadenylate synthetase (2959AS) enzyme activity is controlled by a..., Bonnevie-Nielsen [/bib_ref]. ## Generation of simulated expression dataset A series of simulated expression datasets were generated to contain a total of 20,000 ''genes'' (or probes) and 100 samples. The samples were divided into equally sized groups of 50 control samples and 50 experimental samples, with the gene expression values for each group generated from a standard normal distribution (i.e., with a mean value of 0 and standard deviation of 1). In order to simulate example cases of gene outliers a range of genes were randomly selected to contain an equal number (20, 50, 100, 250, 500, and 1000) of up-and down-regulated patient samples relative to the control group. For each of these simulated outlier genes, we randomly assigned a number of samples from the experimental test group (between 5 and 10) to correspond to expression levels significantly different to the standard normal distributions. These expression levels were randomly assigned so as to correspond to p-values of being part of the standardised normal sample distributions of between 0.05 and 1610 26 . # Results ## Comparison to existing gene outlier detection methods Recently, the GTI outlier detection algorithm was described and compared to other existing gene expression outlier detection methods [bib_ref] GTI: a novel algorithm for identifying outlier gene expression profiles from integrated..., Mpindi [/bib_ref]. It was shown that the GTI method performs comparably to other outlier detection methods (including: cancer outlier profile analysis (COPA); outlier sums (OS); and the outlier robust t-statistic (ORT)) when using a simulated expression dataset [bib_ref] Cancer outlier differential gene expression detection, Wu [/bib_ref] [bib_ref] COPA-cancer outlier profile analysis, Macdonald [/bib_ref] [bib_ref] Outlier sums for differential gene expression analysis, Tibshirani [/bib_ref]. In order to benchmark our z-score based outlier detection method ZODET against these existing methods we performed a similar comparison to the GTI method, using both simulated expression datasets and also an expression dataset compiled from a cohort of human monocyte-derived macrophage samples split into two equal sized groups. Both the ZODET and the GTI methods were run on the simulated expression datasets of 100 samples and 20,000 probes and the resulting outputs compared. ZODET was run with the default p-value detection method and threshold, of 0.01, and a fold-change threshold of 0.5. The GTI method was also run with default settings (except that an automatic log transformation of the expression values was not carried out). To compare the ability of the two methods to correctly identify probes that correspond to the simulated outlier expression values we used Receiver Operating Characteristic (ROC) curves on the ranked outputs [fig_ref] Figure 3: Receiver operating characteristic [/fig_ref]. The GTI output was ranked in descending ''GTI score'' order and ZODET was ranked using the number of samples that were detected to be associated with an outlier probe. These methods of ranking highlight an important difference between outlier detection using ZODET and the GTI method; that is, the GTI method only detects whether a particular gene (or microarray probe) contains outlier samples, whereas ZODET detects the likelihood that a gene (or probe) is an outlier for each of the individual test samples. Therefore, ZODET has the advantage of being able to identify both the genes and the specific test experimental samples that are significantly associated with outlier gene expression values. Interestingly, both methods consistently performed well when identifying the up-regulated true positive outliers in the simulated expression dataset, resulting in an area under the ROC curve (AUC) of 1.0 [fig_ref] Figure 3: Receiver operating characteristic [/fig_ref]. When identifying the down-regulated outliers, ZODET, clearly out-performed the GTI method, with average AUC values of 0.998 and 0.476, respectively [fig_ref] Figure 3: Receiver operating characteristic [/fig_ref]. This was expected because the GTI method is specifically designed to identify outliers that are over-expressed relative to the control group, whereas ZODET detects both under-and over-expressed outliers equally well. In summary, the two methods performed equivalently when identifying the up-regulated outlier genes from the simulated dataset, but importantly ZODET is also able to detect downregulated outliers to a high level of accuracy. ## Identification of outlier genes in a monocyte-derived macrophage microarray dataset An example microarray dataset is available on our webpage (http://www.ucl.ac.uk/medicine/molecular-medicine/publications/ microarray-outlier-analysis). This dataset consists of two data files: (i) an ''EXPRESSION DATA'' file -containing the microarray expression data from monocyte-derived macrophages collected from 40 volunteers split into two equal sized groups labeled control (A1 to 20) and experimental (B1 to 20); and (ii) an ''EXPERIMENT INFORMATION'' file -containing associated experimental parameters. The technical specifications of these input files are described in the provided software documentation. A ZODET analysis was performed with the thresholds set at a log2 fold-change .1.75 and unadjusted p-value ,0.0001. Combining the ZODET analysis results from subjects B1 to B20 identified 19 up-and 14 down-regulated probes in this population [fig_ref] 1: Test Individual vs Control Group 2 [/fig_ref]. Three probes corresponding to two genes were identified as significantly up-regulated in three or more individuals within the experimental cohort. Three individuals were found to over-express either G-protein coupled receptor 128 (GPR128) (ILMN_2125395 and ILMN_1808078) or chemokine (C-C motif) ligand 3-like 1 (CCL3L1)(ILMN_1773245). Two probes corresponding to two genes were found to be down-regulated in three or more individuals in the experimental population. 29-59oligoadenylate synthetase 1 (OAS1) (ILMN_1658247) and ribosomal protein S23 (RPS23) (ILMN_1772459) were significantly attenuated in six and three of the twenty subjects tested, respectively. However, the probe for RPS23 (ILNM_1772459) contains a recognised single nucleotide polymorphism (SNP) (rs3738) which accounts for the three down-regulated outliers identified in the experimental cohort. The SNP results in poor probe hybridisation and not a genuine reduction in gene expression. PCR verification should be conducted on all identified probes in order to discount false positives resulting from poor probe hybridisation. A GTI analysis was also performed on the same data set and the results compared to the output from ZODET [fig_ref] 1: Test Individual vs Control Group 2 [/fig_ref]. In accordance with the simulated data analysis, the GTI software was only capable of identifying the up-regulated probes and gave the highest ranking to both of the GPR128 probes which were also identified using ZODET. In order to determine if the abnormal gene expression results from genetic mutations, both GPR128 and OAS1 genes were sequenced in the subjects identified as outliers. No mutations were found in any of the individuals who over-expressed GPR128 (data not shown). However, a previous report identified a chromosome translocation involving GPR128 and TRK-fused gene (TFG) which results in the generation of an in-frame TFG-GPR128 fusion transcript [bib_ref] TFG, a target of chromosome translocations in lymphoma and soft tissue tumors,..., Chase [/bib_ref]. This transcript was found to be constitutively expressed in all tissues and could be the possible cause of GPR128 over expression identified in these individuals. PCR analysis and sequencing was performed on the three outlier subjects . The results confirmed the presence of the TFG-GPR128 translocation in the three patients identified as outliers for the GPR128 gene. Further PCR analysis was carried out on the remaining 17 experimental subjects and 20 controls. One additional experimental subject was found to carry the translocation and from the expression data the increased levels of GPR128 can be seen but the thresholds applied to the ZODET analysis meant that this individual was not identified as an outlier . None of the control subjects carried the translocation. Therefore the ZODET analysis identified three out of the four experimental subjects that were carriers of the TFG-GPR128 translocation. The most common under-expressed outlier gene was OAS1 which was found in six experimental individuals. The gene was sequenced in all six subjects and was found to be homozygous for a known A/G single-nucleotide polymorphism at the exon 7 splice-acceptor site (rs10774671) in the OAS1 gene [bib_ref] Variation in antiviral 29,59-oligoadenylate synthetase (2959AS) enzyme activity is controlled by a..., Bonnevie-Nielsen [/bib_ref]. Further sequencing of the remaining experimental subjects identified an additional individual who was homozygous for the rs10774671 polymorphism . Therefore, the ZODET analysis identified six of the seven individuals within the experimental cohort that expressed an OAS1 splice variant. In summary, the ZODET analysis identified a number of outlier genes within the experimental population two of which we were able to show resulted from a genetic abnormality. GTI identified the translocation event but missed the splice site polymorphism which resulted in attenuated gene expression and was present in 40% of the experimental population. As these abnormalities were only present in a subset of the patients in the experimental group, standard group-based statistics (e.g. a t-test) would fail to identify a significant difference. These findings demonstrate a potential advantage of the ZODET analysis over the currently available software. # Discussion We have described software designed for the identification, analysis and visualisation of outlier genes and individual outlier samples in microarray expression data via a user-friendly GUI. This method implements a z-score based measure of the level of deviation of individual microarray samples, for a particular gene, from an assumed normal distribution of control comparison samples. This differs in approach from the GTI method, which calculates a score for each gene related to the proportion of samples in a specific group that have gene expression levels above a defined statistical outlier threshold. An important advantage of the ZODET software is the user-friendly implementation of a zscore based outlier detection method, which has been specifically designed to be used by individuals with limited bioinformatic experience through a GUI. Since the GTI method was previously shown to perform comparably to other outlier detection methods we benchmarked the performance of ZODET to this method [bib_ref] GTI: a novel algorithm for identifying outlier gene expression profiles from integrated..., Mpindi [/bib_ref]. We have shown that ZODET performs comparably to the GTI method when identifying up-regulated outliers in a large simulated expression dataset containing a defined numbers of outliers at varying levels of statistical significance from the standardised population mean. ZODET was also shown to perform with a very high accuracy and very low false positive rate when identifying genes and samples simulated to represent down-regulated outliers. In addition to outlier analysis, other methods that use gene expression variability between groups can be used to identify genes that may have biological relevance [bib_ref] Differential variability analysis of gene expression and its application to human diseases, Ho [/bib_ref] [bib_ref] Genetic variants contribute to gene expression variability in humans, Hulse [/bib_ref] [bib_ref] Gene expression variability within and between human populations and implications toward disease..., Li [/bib_ref] [bib_ref] Variance of gene expression identifies altered network constraints in neurological disease, Mar [/bib_ref]. However, in general for these approaches a more robust and dynamic change in expression is required, whereas outlier analysis methods, such as ZODET, can identify an individual or small sub-groups within the experimental population. We have also demonstrated the power of the method for detecting outlier genes of biological interest in a dataset containing gene expression profiles from a cohort of primary monocyte derived macrophages isolated from human subjects. In particular, the most common under-and over-expressed genes identified by ZODET were shown to result from germline mutations. A functional consequence resulting from the TFG-GPR128 translocation has so far not been reported. Whereas, a previous report has shown that the OAS1 splice site mutation results in the loss of the commonly expressed OAS1 enzyme (p46) and the generation The GTI ranking is based on the 22,375 probes analysed. ZODET thresholds were set at p,0.0001 and fold change .1.75. All identified probes are shown along with, the gene symbol, the number of individuals who were classified as outliers, the GTI score and GTI rank. The ''count'' shows the number of individuals from the experimental group that were classified as outliers with respect to the control group for each probe. The 19 up-regulated probes and 14 down-regulated probes are shown. doi:10.1371/journal.pone.0081123.t001 of a dual-function antiviral/proapoptotic isoform (p48) and a novel isoform (p52) with no known role. [bib_ref] Variation in antiviral 29,59-oligoadenylate synthetase (2959AS) enzyme activity is controlled by a..., Bonnevie-Nielsen [/bib_ref] The fact that these genetic alterations can be identified provides evidence that this form of analysis could be of benefit in the study of complex heterogeneous diseases. Proof that this form of analysis can provide diseaserelevant findings comes from the identification of the fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer through the use of the COPA software [bib_ref] Recurrent fusion of TMPRSS2 and ETS transcription factor genes in prostate cancer, Tomlins [/bib_ref]. Of particular note was the ability of ZODET to identify outlier genes that were either significantly up-or down-regulated when compared to the controls. ZODET is also capable of identifying an individual gene expression abnormality in one experimental subject, which may allow the identification of rare genetic events that have extremely high disease penetrance. These properties provide an important addition to other commonly used outlier detection methods, such as COPA and GTI, which concentrate solely on the identification of strongly up-regulated or amplified genes in subgroups of patients. Each of these outlier detection methods has advantages and disadvantages and they could well be most effectively utilised in combination with each other, although we have not attempted to do so in this study. [fig] N: Scatter plot of the correlation between the individual and mean control group expression values, with under and over expressed outlier probes colored green and red respectively (Figure 2B); Volcano plot (Figure 2B) highlighting the detected outlier probes: with fold-change and 2log 10 (p-value) on the horizontal and vertical axes, respectively. Under and over expressed outlier-probes are colored green and red respectively; Heatmap of outlier probes for each individual (Figure 2B): the probes are clustered on the outlier expression data, using hierarchical clustering in the heatmap.2 function from the gplots R package (http://CRA.R-project.org/package = gplots); Gene list tables: containing expression values, probe IDs and gene names of the identified outliers.For the experimental group (Figure 2C): Combined heat-maps: containing all identified outliers; Combined gene list summary tables: containing all identified outliers showing which individual samples the specific outlier was detected in and thus allowing identification of individuals with common outlier probes. [/fig] [fig] Figure 3: Receiver operating characteristic (ROC) curves for ZODET and GTI outlier detection. ROC curves are plotted based on simulated outliers (for a range of up-and down-regulated probes) from a dataset of 20,000 probes. doi:10.1371/journal.pone.0081123.g003 [/fig] [table] 1: Test Individual vs Control Group 2. Test Individual vs All Samples 3. Test Individual vs Test Group 4. Control Individual vs Control Group 5. Control Individual vs All Samples [/table]

Acute acalculous cholecystitis in hospitalized patients in intensive care unit: study of 5 cases # Introduction Acute acalculous cholecystitis (AAC) is an uncommon condition characterized by gallbladder inflammation in the absence of biliary calculi or sludge [bib_ref] The increasing prevalence of acalculous cholecystitis in outpatients. Results of a 7-year..., Savoca [/bib_ref]. It represents 2%-14% of cases of acute cholecystitis [bib_ref] Acalculous cholecystitis: is an elective interval cholecystectomy necessary, Abbas [/bib_ref] [bib_ref] Acute acalculous cholecystitis, Barie [/bib_ref] , with a higher prevalence among males. In spite of AAC can be diagnosed in patients without previous or concomitant comorbidities, it is usually associated with an underlying medical comorbidity [bib_ref] Acute acalculous cholecystitis, Barie [/bib_ref]. Several predisposing factors including gallbladder epithelial ischemia and reperfusion injury, positive pressure ventilation, parenteral nutrition, and opioid use have been implicated [bib_ref] Gallbladder disease in patients on long-term parenteral nutrition, Roslyn [/bib_ref] [bib_ref] Experimental study on the pathogenesis of acute acalculous cholecystitis wsrttromd, free radicals..., Taoka [/bib_ref]. Although the precise mechanism is unknown, the most commonly postulated theories regarding its pathogenesis are bile stasis, sepsis, and ischemia of the gallbladder wall. Bile stasis may result in alterations of change in the chemical composition of bile, subsequently rendering the gallbladder mucosa susceptible to local injury. The role of ischemia in this process has been difficult to elucidate and etiologies of ischemia are multifactorial [bib_ref] Risk factors and therapeutic outcomes of acute acalculous cholecystitis, Gu [/bib_ref]. Due to its non-specific presentation, diagnosis of AAC is challenging, in fact symptoms and laboratory findings are unspecific and are likely to be masked in patients with critically ill diseases or neurological problem. AAC can be diagnosed in children too, mainly associated to viral infections such as Epstein-Barr virus (EBV) and, thus, often associated to liver dysfunction as well. Early detection and timely treatment are advocated in order to avoid a fulminant course of the disease and potential life threatening complications such as gangrene or perforation [bib_ref] Risk factors and therapeutic outcomes of acute acalculous cholecystitis, Gu [/bib_ref] [bib_ref] Acalculous cholecystitis in severely burned patients: incidence and predisposing factors, Theodorou [/bib_ref]. In fact, Prognosis may depends on prompt diagnosis and active treatment [bib_ref] Clinical characteristics of patients with newly developed acute cholecystitis after admission to..., Kim [/bib_ref]. Mortality rates may reach 90% in some study [bib_ref] Emergent cholecystostomy is superior to open cholecystectomy in extremely ill patients with..., Simorov [/bib_ref] [bib_ref] Recent advances in management of acalculous cholecystitis, B B [/bib_ref] [bib_ref] Is cholecystectomy the treatment of choice for acute acalculous cholecystitis? A systematic..., Soria Aledo [/bib_ref]. For patients with evidence of gallbladder gangrene or perforation, cholecystectomy is indicated as an aggressive immediate procedure to improve multi-organ failure [bib_ref] Syrj€ al€ a, T.I. Ala-Kokko, Organ system dysfunction following open cholecystectomy for..., Laurila [/bib_ref]. For critically ill patients or those who are unfit for surgery, percutaneous cholecystostomy (PC) is recommended [bib_ref] Acalculous cholecystitis: is an elective interval cholecystectomy necessary, Abbas [/bib_ref] [bib_ref] Percutaneous cholecystostomy in critically ill patients with acute cholecystitis: complications and late..., Atar [/bib_ref] [bib_ref] A nationwide examination of outcomes of percutaneous cholecystostomy compared with cholecystectomy for..., Anderson [/bib_ref]. # Methods This is a retrospective, descriptive multiple cases study conducted in the Medical Intensive Care Unit of Tahar Sfar Hospital of Mahdia over a 4-year period extending from January 2016 to December 2019. AAC was defined as acute cholecystitis (AC) in the absence of gallstones in the gallbladder or in the main bile duct on abdominal imaging studies or in Intraoperative Cholangiogram. A retrospective review of cases from our healthcare facility in this period was carried out. Demographic data, risk factors, clinical signs, complementary tests, intraoperative findings, and long-term consultation follow-up were collected. Patients included in this study presented cholecystitis that acalculous character was confirmed during surgery after opening the gallbladder and sieving the bile. We excluded all patients with calculous cholecystitis or acalculous cholecystitis but with a main bile duct stone in Intraoperative Cholangiogram reflecting calcific migration from the gallbladder. AAC was diagnosed using abdominal sonography or computed tomography. Imaging studies were interpreted by an expert radiologist. All cases were confirmed by board-certified gastroenterologists or general surgeons based on clinical features and imaging findings. The date of diagnosis of AAC was defined as the day of abdominal imaging. Demographic and clinical data were collected from the patients' medical records. Variables such as age, sex, comorbidities including diabetes and cardiovascular disease, Charlson comorbidity index (CCI), cause of admission, Sequential Organ Failure Assessment (SOFA) score, interval from admission to diagnosis of AAC, treatment for AAC, histological findings, bacterial diagnosis, length of hospital and ICU stays, and mortality were collected. Additionally, red blood cell transfusion history, prior vasoactive drug, opioid use, total parenteral nutrition, and mechanical ventilation, which are known as a risk factors of AAC, were also collected. Statistical analysis: Owing to the small number of patients, data are presented with descriptive statistics measures. Descriptive parameters were expressed as mean (AE standard deviation). The different binary variables were expressed in number of cases. The ethics comity approval was obtained (name of the ethics committee: ethics comity of Tahar Sfar Hospital, Institution: faculty of medicine of Monastir, Certificate number: CEM-2021-11-04). Informed consent was obtained from all subjects and/or their legal guardian(s). # Results In this period 148 patients were assessed for acute calculous or acalculous cholecystitis, the prevalence of AAC among all the patients developing acute cholecystitis is 3%. Five patients were diagnosed with AAC after ICU admission during the study period. The patients' mean age was 62.5 years. All patients were older than 50 years. Patients details are reported in [fig_ref] Table 1: Baseline characteristics of the patients [/fig_ref]. All patients had gallbladder distension with a transverse diameter greater than 5 cm on ultrasound. Thickening of the gallbladder wall greater than 3 mm was found in four patients. The absence of gallstones was reported in all patients. Ultrasound Murphy's sign was present in one patient. Abdominal CT scan was performed in two patients; images showed gallbladder distension greater than 5 cm, gallbladder wall thickening greater than 3 mm and wall enhancement defect without the presence of calculus in these patients. One patient had perivesicular fat infiltration and one patient had perivesicular fluid imaging with subserosal edema. According to Tokyo Guidelines, all patients were classified as Grade 3. Regarding the surgical risk assessment, all patients were classified ASA IV. Probabilistic antibiotic therapy was subsequently documented by an antibiogram performed on a bile sample after cholecystectomy. The combination Imipenem-Tigecycline was used in two patients. The following combinations: Cefotaxime-Metronidazole, Imipenem-Amikacin and Tygecycline-Colistin were used in one patient each. We used the SOFA score to evaluate the evolution of our patients from the day of cholecystectomy to the 7th postoperative day. A decrease in the SOFA score was observed in patients with a favorable postoperative course. The average postoperative length of stay of our patients after cholecystectomy was 18 days AE 12. Only one patient had postoperative peritonitis. Three patients died due to multi-visceral failure [fig_ref] Table 3: Summary of patients with AAC in our study BIS [/fig_ref]. # Discussion The pathogenesis of AAC appears to be multifactorial: bile stasis, sepsis, and ischemia of the gallbladder wall are the most commonly postulated theories, but the precise mechanism is still unclear [bib_ref] Acute acalculous cholecystitis, Barie [/bib_ref]. The gallbladder mucosa is susceptible to local injury when the chemical composition of bile is changed by the bile stasis [bib_ref] Acute acalculous cholecystitis, Barie [/bib_ref]. Bile viscosity increasing intraluminal pressure and Ischemia resulting from hypotension or atherosclerosis has also been implicated as a possible cause. In most cases, hypovolaemia or sepsis are responsible for Gallbladder ischaemia. in fact, mediators released in response to systemic inflammation can induce inflammatory process in the gallbladder [bib_ref] Recent advances in management of acalculous cholecystitis, B B [/bib_ref]. Additionally, in critically ill patients, sepsis and infection are frequently linked to cholestasis [bib_ref] Risk factors and therapeutic outcomes of acute acalculous cholecystitis, Gu [/bib_ref] [bib_ref] Clinical characteristics of patients with newly developed acute cholecystitis after admission to..., Kim [/bib_ref] which is cytokine-mediated, and resulting from reaction to bacterial endotoxins [bib_ref] Risk factors and therapeutic outcomes of acute acalculous cholecystitis, Gu [/bib_ref]. Therefore, cholestasis, gallbladder ischemia, and systemic inflammation caused by pneumonia or any other sepsis could be responsible for the development of AAC in ICU patients [bib_ref] Recent advances in management of acalculous cholecystitis, B B [/bib_ref]. In the series reported by Savoca et al. [bib_ref] The increasing prevalence of acalculous cholecystitis in outpatients. Results of a 7-year..., Savoca [/bib_ref] , underlying medical problems were present in 83% of patients and 72% of them had underlying vascular disease. According to Hakala et al. [bib_ref] Microangiopathy in acute acalculous cholecystitis, Hakala [/bib_ref] microcirculation disturbancy could be an important factor in the pathogenesis of AAC. In fact, when it is compared to acute gallstone-associated cholecystitis, the capillary filling in AAC was poorer and more irregular. Savoca et al. [bib_ref] The increasing prevalence of acalculous cholecystitis in outpatients. Results of a 7-year..., Savoca [/bib_ref] reported 2 cases of AAC without any risk other than severe visceral atherosclerosis, suggesting that impaired mucosal resistancy caused by visceral atherosclerosis could increase risk of AAC. In the present study, all our patients had diseases associated with atherosclerotic vascular problems, such as hypertension, diabetes, cerebro-vascular accidents or coronary artery disease. Some study reported an association between developing AAC and prolonged intensive care length of stay. In fact, Long ICU stay can be a result or a risk factor for developing AAC. In our study the mean length of stay is 9 days, the same results were found by Laurila et al. [bib_ref] Syrj€ al€ a, T.I. Ala-Kokko, Organ system dysfunction following open cholecystectomy for..., Laurila [/bib_ref] : for patients with AAC, the mean length of stay in the ICU was 8 days. This risk depends also on surgery indication; after cardiovascular surgery, the mean length of stay was 26 days [bib_ref] Perioperative predictors of acute cholecystitis after cardiovascular surgery, Rady [/bib_ref]. In trauma patients, it was 13.2 days [bib_ref] Cholecystitis after trauma, Hamp [/bib_ref]. In fact, burns [bib_ref] Acute acalculous cholecystitis in burns: a review, Walsh [/bib_ref] , cardiovascular surgery, or trauma [bib_ref] Acalculous cholecystitis in severely burned patients: incidence and predisposing factors, Theodorou [/bib_ref] [bib_ref] Acute acalculous cholecystitis after trauma: a prospective study, Pelinka [/bib_ref] ,are frequent with those patients. For Laurila et al. [bib_ref] Syrj€ al€ a, T.I. Ala-Kokko, Organ system dysfunction following open cholecystectomy for..., Laurila [/bib_ref] operatively confirmed AAC are more frequent with severe illness, long ICU stay, multiple organ failure and Infection. Prolonged fasting and TPN, prior opioid or prior vasoactive drugs, and mechanical ventilation were frequently observed in patients who developed AAC in the ICU [bib_ref] Clinical characteristics of patients with newly developed acute cholecystitis after admission to..., Kim [/bib_ref]. Clinical characteristics of AAC in the ICU have been reported by some studies but still nonspecific. Due to its variable presentation, diagnosis of AAC is challenging, in fact symptoms and laboratory findings are unspecific. In the present study abdominal pain was noted in only one patient, fever in only two patients. Therefore, the diagnosis of AAC has to be suspected, each time the patient is hospitalized in the intensive care unit for more than one week and has sign of indeterminated infection. In matter of AAC, Ultrasound has high sensitivity and specificity of more than 90% [bib_ref] The diagnosis of acute acalculous cholecystitis: a comparison of sonography, scintigraphy, and..., Mirvis [/bib_ref] , and it can be very useful not only in the early diagnosis of AAC but also in defining the treatment plans to be undertaken [bib_ref] Point-of-care ultrasound: infection control in the intensive care unit, Lichtenstein [/bib_ref]. Images in ultrasound can be a distended gallbladder, a thickened wall, inflammation and pericholecystic fluid without any stones in the gallbladder [bib_ref] Accuracy of ultrasonography in the diagnosis of acute calculous cholecystitis: review of..., Pinto [/bib_ref]. Abnormal ultrasound findings could also be found in the gallbladder without having acalculous cholecystitis in critically ill patients [bib_ref] Prevalence and significance of gallbladder abnormalities seen on sonography in intensive care..., Boland [/bib_ref] , decreasing the specificity of ultrasounds. Computed tomography (CT) imaging has the same sensitivity of ultrasound but lacks of specificity. Critically ill patients have a higher frequency of gallbladder abnormalities on CT compared with ultrasound. In a retrospective study accounting 127 acalculous cholecystitis [bib_ref] Diagnostic accuracy of computed tomography imaging of surgically treated acute acalculous cholecystitis..., Ahvenjarvi [/bib_ref] , 96% of critically ill patients had abnormal CT images: increased thickness, lack of enhancement of the gallbladder wall, subserosal edema, increased bile density, large perpendicular diameters of the gallbladder, gas within the gallbladder, ascites, peritoneal fat edema, and diffuse tissue edema. Finding gas in the gallbladder was the most specific finding for acalculous cholecystitis with specificity of 99.2% but a very low sensitivity of 11.1%. CT scan has a very good negative predictive value, effectively ruling out acalculous cholecystitis. Imaging criteria for the diagnosis of acalculous cholecystitis have been proposed in many study, such as a review from Barie and Eachempati [bib_ref] Acute acalculous cholecystitis, Barie [/bib_ref]. The main CT images to search for are a distended gallbladder without any stones with a thickened or edematous wall. Cholecystectomy is the treatment of choice for AAC. However, surgery is not always possible, in fact it depends on the patient's condition especially that many of these patients are poor surgical candidates. For those patients, a PC tube placed by interventional radiology to secure gallbladder drainage can be used. This can be a temporary solution or a treatment option. As it has been suggested by a large retrospective study, for selected patients, PC can have a lower morbidity, fewer intensive care unit admissions, a decreased length of stay, and lower costs compared with open cholecystectomy [bib_ref] Emergent cholecystostomy is superior to open cholecystectomy in extremely ill patients with..., Simorov [/bib_ref] with a low overall rate of complications (around 2%) [bib_ref] Role of percutaneous cholecystostomy for acute acalculous cholecystitis: clinical outcomes of 271..., Noh [/bib_ref]. In addition, higher mortality rates could be linked to their conditions and not directly to the PC. Transpapillary drainage through an endoscopic retrograde cholangiopancreatography (ERCP) has been used by some authors. Its success was variable and is associated with high recurrence rates [bib_ref] Emergent cholecystostomy is superior to open cholecystectomy in extremely ill patients with..., Simorov [/bib_ref]. The findings of this study must be seen in light of some limitations. First, it is a retrospective single-center study with a small number of patients included, so it may not reflect all critically ill patients in various hospital settings and second, the prevalence of AAC might be underestimated in our study. Larger prospective studies are recommended to assess more strong results. # Conclusion acalculous cholecystitis has non-specific presentation, its Diagnosis is challenging. Symptoms and laboratory findings are unspecific and there are no clear diagnostic criteria. Early diagnosis and timely treatment are very important in order to avoid complication such as perforation and sepsis. In critical ill patient, the diagnosis of AAC should be advoked each time there is signs of infection and after eliminating common causes. Ultrasounds and CT scan can be of a great help. A distended gallbladder, a thickened wall, inflammation and pericholecystic fluid can be found. Gas in the gallbladder is the most specific finding for acalculous cholecystitis. For patients with evidence of gallbladder gangrene or perforation, Cholecystectomy is the treatment of choice for AAC as an aggressive immediate procedure [bib_ref] Clinical characteristics of patients with newly developed acute cholecystitis after admission to..., Kim [/bib_ref]. For critically ill patients or those who are unfit for surgery, percutaneous cholecystostomy (PC) is recommended [bib_ref] Acalculous cholecystitis: is an elective interval cholecystectomy necessary, Abbas [/bib_ref] [bib_ref] Cholecystitis after trauma, Hamp [/bib_ref] [bib_ref] Acute acalculous cholecystitis in burns: a review, Walsh [/bib_ref]. ## Ethics approval and consent to participate we have the approval of the ethics committee. All methods were performed in accordance with the relevant guidelines and regulations and have been performed in accordance with the Declaration of Helsinki. ## Ethics comity approval Name of the ethics committee: ethics comity of Tahar Sfar Hospital. Institution: faculty of medicine of monastir. Certificate number: CEM-2021-11-04. ## Consent for publication Consent from all subjects and/or their legal guardian(s) for publication of identifying information/images in an online open-access publication. # Declarations # Author contribution statement All authors listed have significantly contributed to the investigation, development and writing of this article. # Funding statement This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. # Data availability statement Data included in article/supp. material/referenced in article. ## Declaration of interests statement The authors declare no conflict of interest. ## Additional information Supplementary content related to this article has been published online at https://doi.org/10.1016/j.heliyon.2022.e11524. [table] Table 1: Baseline characteristics of the patients (n ¼ 5). [/table] [table] Table 3: Summary of patients with AAC in our study BIS: biological inflammatory syndrome, OC: open cholecystectomy. [/table]

Assessing the Accuracy of Inhomogeneous Fluid Solvation Theory in Predicting Hydration Free Energies of Simple Solutes Accurate prediction of hydration free energies is a key objective of any free energy method that is applied to modeling and understanding interactions in the aqueous phase. Inhomogeneous fluid solvation theory (IFST) is a statistical mechanical method for calculating solvation free energies by quantifying the effect of a solute acting as a perturbation to bulk water. IFST has found wide application in understanding hydration phenomena in biological systems, but quantitative applications have not been comprehensively assessed. In this study, we report the hydration free energies of six simple solutes calculated using IFST and independently using free energy perturbation (FEP). This facilitates a validation of IFST that is independent of the accuracy of the force field. The results demonstrate that IFST shows good agreement with FEP, with an R 2 coefficient of determination of 0.99 and a mean unsigned difference of 0.7 kcal/mol. However, sampling is a major issue that plagues IFST calculations and the results suggest that a histogram method may require prohibitively long simulations to achieve convergence of the entropies, for bin sizes which effectively capture the underlying probability distributions. Results also highlight the sensitivity of IFST to the reference interaction energy of a water molecule in bulk, with a difference of 0.01 kcal/mol changing the predicted hydration free energies by approximately 2.4 kcal/mol for the systems studied here. One of the major advantages of IFST over perturbation methods such as FEP is that the systems are spatially decomposed to consider the contribution of specific regions to the total solvation free energies. Visualizing these contributions can yield detailed insights into solvation thermodynamics. An insight from this work is the identification and explanation of regions with unfavorable free energy density relative to bulk water. These regions contribute unfavorably to the hydration free energy. Further work is necessary before IFST can be extended to yield accurate predictions of binding free energies, but the work presented here demonstrates its potential.k is Boltzmann's constant, ρ°is the number density of bulk water, and Ω is the integral over the angles. For the TIP4P-2005 water model, ρ°is calculated from a 100 ns NPT simulation of bulk water at 300 K and 1 atm as 0.0331 707 molecules/Å 3 . The solute−water orientational correlation functions were calculated using Euler angles by computing α, cos β, and γ in a fixed reference frame. The limits of integration for the water molecule are between 0 and 2π for α, between −1 and 1 for cos β, and between 0 and π for γ, due to water's C 2v # ■ introduction Inhomogeneous fluid solvation theory 1 (IFST) is a statistical mechanical framework for calculating the effect of a solute on the free energy of the surrounding solvent relative to its bulk state. [bib_ref] Inhomogeneous Fluid Approach to Solvation Thermodynamics. 2. Applications to simple fluids, Lazaridis [/bib_ref] The solute can be a protein, [bib_ref] Thermodynamics of Buried Water Clusters at a Protein-Ligand Binding Interface, Li [/bib_ref] peptide, [bib_ref] Structural Properties of Hydration Shell Around Various Conformations of Simple Polypeptides, Czapiewski [/bib_ref] or small molecule 5 and the solvent is commonly water. [bib_ref] Entropy-Based Measure of Structural Order in Water, Esposito [/bib_ref] IFST has found particular use in the pharmaceutical industry with the advent of Schrodinger's WaterMap software. [bib_ref] Motifs for Molecular Recognition Exploiting Hydrophobic Enclosure in Protein-Ligand Binding, Young [/bib_ref] It has also been developed in the solvation theory of ordered water (STOW) package, [bib_ref] Computing the Thermodynamic Contributions of Interfacial Water, Li [/bib_ref] and recent work using grid inhomogeneous solvation theory (GIST) has explored the results of performing IFST calculations on a Cartesian grid. [bib_ref] Grid Inhomogeneous Solvation Theory: Hydration Structure and Thermodynamics of the Miniature Receptor..., Nguyen [/bib_ref] One of the useful features of IFST is that the free energy changes are calculated for small subvolumes surrounding the solute and this allows the contribution of different regions of space to be calculated and visualized. This has been used to understand the determinants of binding affinity [bib_ref] High-Energy Water Sites Determine Peptide Binding Affinity and Specificity of PDZ Domains, Beuming [/bib_ref] and design new inhibitors in the hit-to-lead and lead optimization stages of drug development. [bib_ref] Contribution of Explicit Solvent Effects to the Binding Affinity of Small-Molecule Inhibitors..., Abel [/bib_ref] Work in this lab has focused on the importance of modeling solvation at protein surfaces [bib_ref] Thermodynamic Properties of Water Molecules at a Protein Protein Interaction Surface, Huggins [/bib_ref] and the data requirements for convergence of the thermodynamic quantities computed by IFST. [bib_ref] Benchmarking the Thermodynamic Analysis of Water Molecules Around a Model Beta Sheet, Huggins [/bib_ref] One important issue that has not been addressed in detail is the quantitative accuracy of IFST. Initial work suggested a reasonable comparison with the experimental solvation free energy of methane [bib_ref] Solvent Reorganization Energy and Entropy in Hydrophobic Hydration, Lazaridis [/bib_ref] and recent work notes solvation free energies as a key benchmark for the method. [bib_ref] Grid Inhomogeneous Solvation Theory: Hydration Structure and Thermodynamics of the Miniature Receptor..., Nguyen [/bib_ref] While a direct comparison with experiment is interesting, the results rely on the force field and particularly the water model that is used. [bib_ref] Hydration Thermodynamic Properties of Amino Acid Analogues: A Systematic Comparison of Biomolecular..., Hess [/bib_ref] Thus, a more useful comparison is with an equivalent computational technique. This decouples testing of the method from testing of the parameters. In this work, we compute solvation free energies for six simple molecules using IFST and compare the results with solvation free energies calculated using free energy perturbation (FEP). [bib_ref] High-Temperature Equation of State by a Perturbation Method. I. Nonpolar Gases, Zwanzig [/bib_ref] ## ■ simulation details Hydration free energies for six simple molecule were calculated using two statistical mechanical computational methods, FEP and IFST. FEP calculates the hydration free energies directly, whereas IFST calculates the hydration enthalpies and entropies and these are combined to yield the predicted hydration free energy. All molecular dynamics (MD) simulations in this work were performed with the TIP4P-2005 water model. [bib_ref] A General Purpose Model for the Condensed Phases of Water: TIP4P, Abascal [/bib_ref] Systems Setup. The six solutes studied were acetamide, benzene, isobutane, methane, methanol, and N-methylacetamide. The parameters for acetamide, benzene, isobutane, methanol, and N-methylacetamide were taken from the CHARMM36 force field. [bib_ref] Update of the CHARMM All-Atom Additive Force Field for Lipids: Validation on..., Klauda [/bib_ref] The parameters for methane were not present in the CHARMM36 force field and were thus adapted from ethane. The methane carbon atom was assigned an atom type of CG331 and a partial charge of −0.36. The methane hydrogen atoms were assigned an atom type of HGA3 and a partial charge of +0.09. The bond lengths, bond angles, and dihedral angles were set to their force field equilibrium values for all molecules. Water Setup. To generate a reasonable initial water density, a water shell of radius 50.0 Å was generated around each biomolecule with the SOLVATE program version 1.0 from the Max Planck Institute.The resulting water globules were then cut to rhombic dodecahedral unit cells with side lengths of 25.0 Å. To standardize the geometries of the water molecules, every hydrogen atom was deleted and all the necessary hydrogen atoms and lone pairs were built using the appropriate geometry for TIP4P-2005 water. The boxes contained 384, 371, 376, 394, 376, and 374 water molecules for the molecules acetamide, benzene, isobutane, methane, methanol, and N-methylacetamide, respectively. No ions were included in the systems. IFST Equilibration. Equilibration was performed for 1.0 ns in an NPT ensemble at 300 K and 1 atm using Langevin temperature control and Nose−Hoover [bib_ref] Constant-Pressure Molecular-Dynamics Algorithms, Martyna [/bib_ref] Langevin piston pressure control. [bib_ref] Constant-Pressure Molecular-Dynamics Simulation -the Langevin Piston Method, Feller [/bib_ref] All systems were brought to equilibrium before continuing, by verifying that the energy fluctuations were stable. MD simulations were performed using an MD time step of 2.0 fs. Electrostatic interactions were modeled with a uniform dielectric and a dielectric constant of 1.0 throughout the equilibration and production runs. van der Waals interactions were truncated at 11.0 Å with switching from 9.0 Å. Electrostatics were modeled using the particle mesh Ewald method, 21 and the systems were treated using rhombic dodecahedral periodic boundary conditions. All solute atoms were fixed for the entirety of the equilibration and production simulations. IFST Simulation. 100.0 ns of production simulation in an NPT ensemble were performed at 300 K and 1 atm for each system. System snapshots were saved every 10.0 fs, yielding 10 000 000 snapshots in total for each system. MD simulations for IFST were performed using NAMD 22 version 2.8 compiled for use with CUDA-accelerated GPUs. IFST Implementation. An important decision in implementing IFST is the choice of subvolumes over which to perform the calculations. In protein binding sites, water molecules commonly cluster in distinct locations and the concept of a hydration site is useful to calculate contributions to the free energy from spherical regions. In the context of small molecule solvation, the water molecules do not cluster in distinct locations and the concept of a hydration site is not useful. Two approaches have been used to account for this in previous work. In the Lazaridis work on the solvation of methane, 5 the region surrounding the solute was split into subshells at different distances from the origin. For more complex molecules, spherical polar coordinates could usefully be used to further split these subshells. In the recent work on GIST, the region surrounding the solute was split into cubic voxels on a Cartesian grid. [bib_ref] Grid Inhomogeneous Solvation Theory: Hydration Structure and Thermodynamics of the Miniature Receptor..., Nguyen [/bib_ref] This has the advantage that each subvolume has the same volume. There are a number of other possibilities for partitioning a volume into subvolumes and these different methods of honeycombing space are likely to affect the performance of IFST. In this work, we have used a Cartesian grid to honeycomb the volume and we use the term voxel to specify one cubic unit in this space. We consider all voxels within 12.0 Å of the origin in the calculation, where the centroid of each solute lies at the origin. In this study, we have not taken advantage of the symmetry of the solute molecules, which would provide more data, as we are interested in applying IFST to systems that do not have such symmetry in the future. IFST Calculations. IFST calculates the difference in interaction energy (ΔE IFST ) and correlation entropy (ΔS IFST ) between each subvolume (v) and the equivalent number of bulk water molecules (n). These can be termed the local quantities. The contribution of each subvolume to ΔE IFST was calculated from the mean solute−water interaction energy (E sw ), the mean water−water interaction energy (E ww ), and the mean interaction energy of a bulk water molecule (E bulk ) as follows: [formula] ∑ ∑ Δ = + − = +Δ E E E n E E E [ ] [ ] v v IFST sw ww bulk sw ww(1) [/formula] For the TIP4P-2005 water model, E bulk is calculated from a 100 ns NPT simulation of bulk water at 300 K and 1 atm as −11.5813 kcal/mol. In this work, E bulk and E ww are defined as half the interaction energy of one water molecule with all other water molecules. Previous work have defined E bulk and E ww as the total interaction energy of one water molecule with all other water molecules and included factors of one-half in eq 1. [bib_ref] Benchmarking the Thermodynamic Analysis of Water Molecules Around a Model Beta Sheet, Huggins [/bib_ref] [bib_ref] Application of Inhomogeneous Fluid Solvation Theory to Model the Distribution and Thermodynamics..., Huggins [/bib_ref] The two approaches are identical. The difference in correlation entropy is calculated from solute−water entropy (S sw ), the water−water entropy (S ww ), and the entropy of a bulk water molecule (S bulk ) as follows: [formula] ∑ ∑ Δ = + − = +Δ S S S n S S S [ ] [ ] v v IFST sw ww bulk sw ww(2) [/formula] All entropies calculated in this work exclude vibrational entropy changes. For each subvolume, S sw is calculated from the translational and orientational contributions using the translational and orientational correlation functions, g sw (r) and g sw (ω| r). [formula] ∫ ρ = −°S k g r g r r ( ) ln ( ) d sw trans sw sw (3) ∫ ρ ω ω ω = −°Ω | | S k g r g r g r r ( ) ( ) ln ( ) d d sw orient [/formula] sw sw sw [bib_ref] Structural Properties of Hydration Shell Around Various Conformations of Simple Polypeptides, Czapiewski [/bib_ref] symmetry. The orientational correlation functions were assumed to be independent of the position within the hydration sites. The default grid resolution was 0.5 Å and the default angular bin size was 45°. This leads to 8, 4, and 4 angular bins for α, cos β and γ and thus 128 angular bins in total. The ΔS ww term can also be calculated from translational and orientational contributions. A rigorous treatment would use the inhomogeneous water−water pair-correlation functions g ww (r,r′) and g ww (ω,ω′|r,r′) from the system and the homogeneous water− water pair-correlation functions g ww (R) and g ww (ω rel |R) from bulk water. R and ω rel are the distance and relative orientation between two water molecules in bulk water. [formula] ∫ ρ Δ = −°′ ′ ′ − ′ + − − + ′ S k g r g r g r r g r r g r r g R g R g R r r 1 2 ( ) ( )[{ ( ) ( , ) ln ( , )( , ) [/formula] However, converging calculations of the inhomogeneous water−water pair-correlation functions requires a very large amount of data and thus the Kirkwood superposition approximation (KSA) is used. [bib_ref] Statistical Mechanics of Fluid Mixtures, Kirkwood [/bib_ref] The KSA provides an approximation of a third-order correlation function in terms of lower order correlation functions. In this context, the KSA is applied to the homogeneous triplet solute−water−water correlation function. The inhomogeneous water−water correlation function is then assumed to be independent of the solute−water correlation functions and thus equal to the water−water correlation function in bulk solvent. The KSA is used for both g ww (r,r′) and g ww (ω,ω′|r,r′): [formula] ′ = g r r g R ( , ) ( ) ww ww bulk (7) ω ω ω ′| ′ = | g r r g R ( , ,) ( ) ww [/formula] These correlation functions can be converged in bulk water due the increased data available from it being a homogeneous and isotropic system. Previous work on the solvation of methane suggests that these KSAs lead to reasonable results due to the cancellation of errors between more and less structured regions. [bib_ref] Solvent Reorganization Energy and Entropy in Hydrophobic Hydration, Lazaridis [/bib_ref] In this work, S ww was only calculated for pairs of voxels within 3.6 Å. This is likely a good assumption for the translational contributions, which in bulk water are derived almost exclusively from the excluded volume and the first solvation shell. [bib_ref] Structural Properties of Water: Comparison of the SPC, SPCE, TIP4P, and TIP5P..., Zielkiewicz [/bib_ref] The orientational contributions to S ww (and S bulk ) are expected to be underestimated, as only approximately 75% of the orientational entropy in bulk water is derived from the first solvation shell. [bib_ref] Structural Properties of Water: Comparison of the SPC, SPCE, TIP4P, and TIP5P..., Zielkiewicz [/bib_ref] [bib_ref] Correlations in Liquid Water for the TIP3P-Ewald, TIP4P-2005, TIP5P-Ewald, and SWM4-NDP Models, Huggins [/bib_ref] Calculations for g ww (R) and g ww (ω rel |R) were performed on pairs of voxels on the Cartesian grid from the 100 ns NPT simulation of bulk water at 300 K and 1 atm. The difference in free energy (ΔG IFST ) for each voxel is then calculated from ΔE and ΔS. [formula] Δ = Δ − Δ G E T S IFST IFST IFST (9) [/formula] FEP Protocol. The equilibrated systems generated for the IFST simulations were used as the start points for the FEP systems. These systems consist of the solute in water and correspond to the λ = 0.0 states. FEP calculations were performed in both forward and backward directions to yield corresponding predictions for annihilation (λ = 0.0 to λ = 1.0) and creation (λ = 1.0 to λ = 0.0) of the solutes. Each annihilation and creation was split into 24 steps to yield 48 λ windows per system. The lambda schedules are reported in [fig_ref] Table 1: Lambda Schedules for FEP Annihilation and Creation a The 24 FEP windows... [/fig_ref]. Two measures were adopted to avoid the numerical instabilities termed "end-point catastrophes" that occur when λ approaches 0.0 or 1.0. First, a soft-core potential was employed with a van der Waals radius-shifting coefficient of 5.0. [bib_ref] Avoiding Singularities and Numerical Instabilities in Free-Energy Calculations Based on Molecular Simulations, Beutler [/bib_ref] [bib_ref] Separation-Shifted Scaling, a New Scaling Method for Lennard-Jones Interactions in Thermodynamic Integration, Zacharias [/bib_ref] Second, van der Waals interactions were scaled down to zero between λ = 0.0 and λ = 1.0 whereas electrostatic interactions were scaled down to zero between λ = 0.0 and λ = 0.4. 29 MD simulations for FEP were performed using NAMD version 2.8. [bib_ref] Scalable Molecular Dynamics with NAMD, Phillips [/bib_ref] FEP Equilibration. Starting with the equilibrated systems generated for the IFST simulations, further equilibration was performed for 800 ps in an NPT ensemble for each lambda window. FEP Simulation. 7.0 ns of production simulation in an NPT ensemble were performed at 300 K and 1 atm for each lambda window. [formula] ∑ Δ = Δ = = + → G G a b a N a b FEP 1, 1(10) [/formula] The free energy change for each small step (ΔG a→b ) is calculated using the partition functions (Q) for the two states which are calculated using the Hamiltonians (H) for the two states. [formula] Δ = − =− = − ⟨ − − ⟩ → ⎛ ⎝ ⎜ ⎜ ⎞ ⎠ ⎟ ⎟ G G G k T Q Q kT H H kT ln ln( exp( ( )/ ) ) a b b a b a b a a(11) [/formula] We employed the Bennett acceptance ratio (BAR) method, 30 a technique that combines forward and backward FEP simulations to provide efficient estimates of free energy changes. [bib_ref] Good Practices in Free-Energy Calculations, Pohorille [/bib_ref] BAR was implemented using the ParseFEP Plugin from VMD. The ParseFEP Plugin is described in "A Toolkit for the Analysis of Free-Energy Perturbation Calculations" and the free energy and the statistical error are computed using eqs 11 and 14 from that paper. [bib_ref] A Toolkit for the Analysis of Free-Energy Perturbation Calculations, Liu [/bib_ref] The estimated statistical error in the FEP free energy predictions using BAR was less than 0.1 kcal/ mol in all cases. Additional Considerations. IFST calculations equate the change in enthalpy (ΔH) with the change in potential energy (ΔE). For calculations in an NPT ensemble, there is an additional contribution to the free energy of PΔV, where P is the pressure and ΔV is the change in volume. However, for solvation of small molecules in water under ambient conditions, PΔVis significantly smaller than ΔE and can be safely neglected. [bib_ref] Enthalpy-Entropy and Cavity Decomposition of Alkane Hydration Free Energies: Numerical Results and..., Gallicchio [/bib_ref] In addition, the FEP and IFST calculations in this study both use a nonpolarizable force field. The parameters for the solutes are for prepolarized molecules and thus the free energy change associated with polarization of the solutes is ignored. [bib_ref] Hydration Thermodynamic Properties of Amino Acid Analogues: A Systematic Comparison of Biomolecular..., Hess [/bib_ref] The calculated values are thus expected to differ from the experimental values. As noted previously, such calculations also ignore the difference in polarization of the water molecules between bulk water and the solution. 14 An additional consideration when comparing IFST calculations to experiment are terms that arise from the thermal motion of the solute and the change in ideal solvent entropy upon solute insertion. The additional terms are commonly termed the liberation contributions (ΔE lib and ΔS lib ) and the volume entropy contribution (S ve ). [bib_ref] Extracting Hydrophobic Free-Energies from Experimental-Data -Relationship to Protein Folding and Theoretical-Models, Sharp [/bib_ref] Collectively, they are termed the nonlocal contributions and are distinct from the correlation terms in eqs 1 and 2.The local contributions ΔE IFST , ΔS IFST , and ΔG IFST represent Ben-Naim's standard energy, entropy, and free energy of solvation. [bib_ref] Standard Thermodynamics of Transfer. Uses and Misuses, Ben-Naim [/bib_ref] The liberation enthalpy ΔE lib is calculated from the solvent's thermal expansion coefficient α and isothermal compressibility κ: [formula] α κ Δ = − E kT T P ( ) lib(12) [/formula] For solvation in water under standard ambient conditions, ΔE lib is approximately +0.05 kcal/mol. IFST is formulated in such a way that portions of ΔS lib and S ve cancel with some of the correlation terms.The entropy from the nonlocal contributions ΔS nonlocal is calculated from the remaining terms: [formula] α Δ = − S kT ( 1) nonlocal(13) [/formula] For solvation in water under standard ambient conditions ΔS nonlocal makes a contribution of +0.55 kcal/mol to the free energy. In this work, we compare the predictions of FEP and IFST with Ben-Naim's experimentally determined standard energy, entropy, and free energy of solvation. These do not include the nonlocal contributions, which are thus excluded from our calculations. [formula] ■ EXPERIMENTAL DATA [/formula] The standard energies, entropies, and free energies of solvation of solvation for benzene, isobutane, methane, and methanol are taken from the same source. [bib_ref] Solvation Thermodynamics of Nonionic Solutes, Ben-Naim [/bib_ref] The standard free energies of solvation for acetamide and N-methylacetamide are derived from Wolfenden [bib_ref] Interaction of the Peptide Bond with Solvent Water: A Vapor Phase Analysis, Wolfenden [/bib_ref] and taken from the same source. [bib_ref] Solvation Free-Energies of Small Amides and Amines from Molecular-Dynamics Free-Energy Perturbation Simulations..., Ding [/bib_ref] The enthalpies of solvation for acetamide and N-methylacetamide are taken from the same source [bib_ref] Enthalpies of Solvation for N-Alkylamides in Water and in Carbon-Tetrachloride at 25-Degrees-C, Dellagatta [/bib_ref] and then modified to yield the standard enthalpies of solvation. [bib_ref] Thermodynamic Decomposition of Hydration Free Energies by Computer Simulation: Application to Amines,..., Kubo [/bib_ref] The entropies of solvation were determined from the standard energies and free energies. ## ■ results and discussion The simulation of bulk water is a key part of this work, as ΔE IFST depends critically upon the calculated value of E bulk . This is because the volume of solvent we are considering (voxels within 12.0 Å of the origin) contains approximately 240 water molecules. Thus, a difference of 0.01 kcal/mol in E bulk leads to a difference of 2.4 kcal/mol in ΔE IFST and ΔE FEP . This is significant in the context of predicting solvation free energies. A simple calculation suggests that E bulk must be correctly converged to within 0.0002 kcal/mol for ΔE IFST and ΔE FEP to be correctly calculated to within 0.1 kcal/mol. This extreme dependence means that it is thus vital that the calculation of E bulk is converged. [fig_ref] Figure 1: Convergence of E bulk for a 100 ns simulation of bulk TIP4P-2005... [/fig_ref] shows the convergence of E bulk for the 100.0 ns NPT simulation. After approximately 75 ns, the running average remains within 0.0002 kcal/mol of the final calculated value, yielding a final E bulk of −11.5813 kcal/mol for the TIP4P-2005 water model. The simulation of bulk water also allows calculation of the excess entropy (S bulk ). Numerous works have reported the excess translational entropy of bulk water based on IFST The RDF between 0.0 and 3.6 Å from the bulk water simulation of TIP4P-2005 (blue line) and an "effective RDF" computed for voxels at the given distance from the origin (red line). The effective RDF is calculated by first computing the distance between the origin and the center of each voxel. Voxels at the same distance from the origin are then grouped together. The value of g(r) at each distance is calculated from the average number of water molecules in the group of voxels at that distance and their summed volume. Due to bulk water being isotropic, the excess translational entropy can be calculated from the radial distribution function (RDF). Using the RDF with a radial bin size of 0.1 Å, the excess translational entropy in the first solvation shell (between 0.0 and 3.6 Å) is calculated to be −3.29 cal/mol/K for the TIP4P-2005 water model. This makes a contribution of +0.98 kcal/ mol to the excess free energy. It is interesting to compare this with the translational entropy based on IFST calculations using a Cartesian coordinate system. Using the default grid resolution of 0.5 Å, the excess translational entropy for voxels within the first solvation shell (between 0.0 and 3.6 Å) is calculated to be −2.10 cal/mol/K. This makes a contribution of +0.63 kcal/mol to the excess free energy. The true RDF and an "effective RDF" using the Cartesian coordinate system can be seen in [fig_ref] Figure 2: Translational correlation function for TIP4P-2005 between 0 [/fig_ref]. The curve is much flatter for the Cartesian coordinate system, with a significantly reduced contribution from the excluded volume. This suggests that a Cartesian grid of 0.5 Å may be too coarse to fully capture the translation probability density function. However, similar results are observed for the solute water RDFs (data not shown) and the errors in the translational entropy for the bulk system and the solute−water systems largely cancel. A similar approach can also be taken for the orientational entropy. Using spherical polar coordinates with a radial bin size of 0.1 Å and an angular bin size of 10°, the excess orientational entropy in the first solvation shell (between 0.0 and 3.6 Å) is calculated to be −7.28 cal/mol/K for the TIP4P-2005 water model. This makes a contribution of +2.17 kcal/mol to the excess free energy. For the Cartesian coordinate system using a grid resolution of 0.5 Å and an angular bin size of 10°, the excess orientational entropy in the first solvation shell (between 0.0 and 3.6 Å) is calculated to be −4.70 cal/mol/K. This makes a contribution of +1.40 kcal/mol to the excess free energy. [fig_ref] Figure 3: Cumulative orientational contribution to TS bulk for bulk TIP4P-2005 water [/fig_ref] shows the cumulative orientational contributions to S bulk between 0.0 and 3.6 Å for the radial and Cartesian coordinate calculations. These findings show that S bulk is underestimated using a grid resolution of 0.5 and this suggests that S ww is likely to be misestimated. After calculating the necessary values for the bulk water simulation, we tested the method by using the bulk simulation as a basis for IFST calculations. The IFST quantities were calculated for all voxels within 12.0 Å of the origin. In all the following work, we report the entropic contributions to the free energy (−TΔS IFST ) in kcal/mol rather than the entropies (ΔS IFST ) in cal/mol/K, as this allows a direct comparison with the enthalpic contributions to the free energy. The second column of [fig_ref] Table 2: Results of IFST Calculations on Bulk Water and the Six Solutes a... [/fig_ref] shows the contributions from the different IFST quantities for the bulk water simulation, which should all be zero when there is no perturbation from a solute. It is clear that the orientational contributions to −TS sw are overestimated by this method and this is due to the inherent bias in the histogram method. The overestimate is very small for each voxel (approximately 0.000 015 kcal/mol) but there are approximately 60 000 voxels and this leads to a significant overestimate in total. For this reason, we expect that the orientational contributions to S sw will be overestimated for the solute systems. The other contributions to the free energy are close enough to zero to be acceptable. We then moved on to consider the solute−water systems. A key question that we have previously attempted to address is the size of the solvation shell around a solute that is affected by its presence. [bib_ref] Benchmarking the Thermodynamic Analysis of Water Molecules Around a Model Beta Sheet, Huggins [/bib_ref] [bib_ref] Application of Inhomogeneous Fluid Solvation Theory to Model the Distribution and Thermodynamics..., Huggins [/bib_ref] However, previous studies were incomplete because they considered the per voxel values of ΔE IFST , ΔS IFST , and ΔG IFST with increasing distance from the origin rather than the total ΔE IFST , ΔS IFST , and ΔG IFST . These total values are affected by the volume increasing with the cube of the distance from the origin and are more revealing. We have calculated these values for the six solutes to more thoroughly address this issue. The results can be seen in [fig_ref] Figure 4: Cumulative contributions to ΔE IFST , ΔS IFST , and ΔG IFST... [/fig_ref] for acetamide. The total ΔS IFST approaches its final value at around 5.0 Å from the origin whereas the total ΔE IFST and thus the total ΔG IFST approach their final value at around 9.0 Å from the origin. The slight peaks in ΔS IFST at 5.0 and 9.0 Å are due to the first and second solvation shells, as the plot is of the distance from the origin and acetamide has an approximate "radius" of 3.0 Å. When the size of the solutes is considered, the results indicate that the solvent is perturbed to a distance of approximately 7.0 Å from the surface of a solute. This corresponds to the first two solvation shells, with the first solvation shell contributing around 80% to ΔG IFST . As discussed previously, these distances may be much larger for charged systems and they may also be different for larger solutes. Indeed, studies on a 16-residue peptide suggest that significant perturbations may extend to the third solvation shell at around 10 Å from the surface. [bib_ref] Local Order, Energy, and Mobility of Water Molecules in the Hydration Shell..., Agarwal [/bib_ref] We also wished to study the convergence of the IFST quantities. As previously, we distinguish between the convergence with increased simulation time for a given sampling frequency and the convergence with increased sampling frequency for a given simulation time. [bib_ref] Benchmarking the Thermodynamic Analysis of Water Molecules Around a Model Beta Sheet, Huggins [/bib_ref] [bib_ref] Application of Inhomogeneous Fluid Solvation Theory to Model the Distribution and Thermodynamics..., Huggins [/bib_ref] The convergence with increased simulation time can be seen for acetamide in [fig_ref] Table 3: Convergence of IFST Predictions with IncreasingSimulation Time a [/fig_ref] for a sampling frequency of 10 fs. The result for ΔE IFST after 20.0 ns differs from the result after 100 ns by only 0.1 kcal/mol but the result for TΔS IFST differs by more than 0.1 kcal/mol, even after 50.0 ns. It is the difference in the orientational component of S sw that is notably different as the simulation time is decreased. This is not unexpected, as there are 128 times as many histogram bins for the orientational component of S sw as for the translational component. These calculations suggest that converged predictions for IFST require at least 100 ns of simulation with a grid resolution of 0.5 Å and an angular bin size of 45°b ut that simulations of 20 ns may be sufficient to predict ΔE IFST . The convergence with increased sampling frequency for acetamide can be seen infor a simulation time of 100 ns. The result for ΔE IFST using a sampling frequency of 1000 fs is the same as the result using a sampling frequency of 10 fs to one decimal place, and the result for TΔS IFST using a sampling frequency of 20 fs is the same as the result using a sampling frequency of 10 fs to one decimal place. Again, it is the orientational component of TS sw that is notably different as the sampling frequency is decreased. These calculations suggest that converged predictions for IFST using a histogram method require at least 5 000 000 samples from a 100 ns simulation with a grid resolution of 0.5 Å and an angular bin size of 45°. Other methods, such as the k-nearest neighbors (k-NN) algorithm, 45 may require shorter simulations and less sampling. [bib_ref] Grid Inhomogeneous Solvation Theory: Hydration Structure and Thermodynamics of the Miniature Receptor..., Nguyen [/bib_ref] [bib_ref] Thermodynamic Properties of Liquid Aater: An Application of a Nonparametric Approach to..., Wang [/bib_ref] We also wished to consider the effect of the grid resolution and angular bin size on the orientational and translational components of S sw . The results in [fig_ref] Table 3: Convergence of IFST Predictions with IncreasingSimulation Time a [/fig_ref] indicate that the translational component of S sw is well converged for a grid resolution of 0.5 Å. We also studied the results for grid resolutions of 0.25 and 0.75 Å to assess whether the translational component of S sw is fully quantified with the default grid resolution of 0.5 Å. The results for acetamide are reported in [fig_ref] Table 5: Effect of the Grid Resolution on the Translational Component of −TS sw [/fig_ref] and indicate that the answer is no. A grid resolution of 0.25 Å yields a converged value of 4.3 kcal/mol for the translational component of −TS sw in comparison with a value of 3.7 kcal/mol for a grid resolution of 0.5 Å. Smaller grid resolutions, which may yield a higher prediction, were not tested. Testing the orientational component of −TS sw in this fashion is more complicated because it requires a grid resolution in addition to an angular bin size. In this work, we have only explored the variation in entropy with respect to the angular bin size at the default grid resolution of 0.5 Å. We studied the results for angular bin sizes of 36°and 60°to assess whether the translational component of −TS sw is fully quantified with the default angular bin size of 45°. The results for acetamide are reported in [fig_ref] Table 6: Effect of the Angular Bin Size on the Orientational Component of −TS... [/fig_ref]. An angular bin size of 36°yields a value of 3.6 kcal/mol for the orientational component of −TS sw in comparison with a value of 2.9 kcal/ mol for an angular bin size of 45°. However, it is not clear that the results for an angular bin size of 36°are fully converged from 10 000 000 samples over 100 ns and thus the question cannot be answered. Indeed, the results from the simulation of bulk water presented in the first column of [fig_ref] Table 2: Results of IFST Calculations on Bulk Water and the Six Solutes a... [/fig_ref] suggest that the orientational component of −TS sw is not fully converged from 10 000 000 samples over 100 ns for an angular bin size of 45°. Due to computational restrictions, we have not tested effect of the grid resolution and angular bin size on the orientational and translational components of ΔS ww . However, the results from the simulation of bulk water suggest that the effects on S sw and ΔS ww may counterbalance one another. The IFST predictions can be seen in [fig_ref] Table 2: Results of IFST Calculations on Bulk Water and the Six Solutes a... [/fig_ref]. Calculations suggest that the translational and orientational contributions to the solvation entropy are of a similar magnitude in all cases, though the orientational contributions are always slightly larger. In addition, the solute−water terms are predicted to be 5−8fold larger than the water−water terms. This is partly because the contribution of the translational water−water term is negative due to the solute excluded volume, but the orientational water−water term is also small. However, there are two issues to be considered. The first is that the water− water terms depend on the validity of the KSAs, which have not been thoroughly explored. The second is that our calculations suggest that the water−water terms are misestimated using a Cartesian grid with a resolution of 0.5 Å. It is interesting to note that the solute−water interactions (E sw ) are highly favorable in all cases, at the expense of an unfavorable change in water− water interactions (ΔE ww ). This leads to a favorable change in overall energy (ΔE IFST ) that is approximately half the magnitude of E sw for the majority of solutes. Assessing the data generated using IFST, it is clear that there are a number of issues with the method. The selection of grid resolution and angular bin size affects all the calculated entropies and in addition the use of the KSAs has not been comprehensively validated. However, these issues affect all solutes and thus we might expect a systematic misestimation of the entropies in comparison with FEP. We proceed, with this in mind. The results from experiment along with the predictions from FEP and IFST can be seen in [fig_ref] Table 7: Results of the FEP and IFST Calculations Compared to Experiment a [/fig_ref]. The correlation plots between experiment, IFST, and FEP can be seen in [fig_ref] Figure 5: Correlation plots between ΔG, ΔH, and TΔS for experiment and the predictions... [/fig_ref]. Of primary interest is the agreement between FEP and IFST. The mean unsigned difference in the solvation free energies is 0.7 kcal/mol, with the IFST predictions tending to be less favorable than the FEP predictions. FEP and IFST both yield reasonable predictions of the experimental quantities with mean unsigned errors for the solvation free energies of 1.3 and 1.8 kcal/mol, respectively. N-Methylacetamide is particularly poorly predicted by both methods. Agreement of IFST with the experimental enthalpic and entropic contributions is reasonable, with mean unsigned errors of 0.9 and 1.2 kcal/ mol, respectively. However, the results suggest that the entropic component −TΔS tends to be overestimated by IFST, with a mean signed error of +1.2 kcal/mol. These results may explain why the IFST predictions of the hydration free energies tend to be less favorable than the FEP predictions. They are also commensurate with the overestimation of TΔS by 1.0 kcal/mol for the simulation of bulk water. One of the main advantages of IFST calculations is the ability to break the solvation free energies into contributions from different regions and visualize these contributions. This promotes understanding of the system being studied and an insight into the thermodynamics of solvation. shows the contributions from different regions to the hydration free energy of acetamide at different contour levels, visualized using VMD. [bib_ref] VMD: Visual Molecular Dynamics, Humphrey [/bib_ref] The region with the highest relative free energy density is a ring surrounding the carbonyl oxygen . The carbonyl group is actually predicted to contribute approximately −6.0 kcal/mol to the hydration free energy of acetamide . At lower contour levels, the contributions from the amide hydrogens are visible, with each a The orientational component of −TS sw for acetamide, calculated from 1 000 000, 2 000 000, 5 000 000, and 10 000 000 samples with angular bin sizes of 30°, 45°, and 60°. contributing approximately −1.5 kcal/mol to the hydration free energy . It is worth noting that the favorable regions surrounding the amide hydrogens would be well described by spherical hydration sites but the favorable regions surrounding the carbonyl oxygen would not. Contouring from positive values, the regions contributing most unfavorably to the hydration free energy are above and below the plain of the amide bond . This effect has been noted in previous studies using WaterMap [bib_ref] Thermodynamic Analysis of Water Molecules at the Surface of Proteins and Applications..., Beuming [/bib_ref] and agrees with observations from the Cambridge Crystallographic Data Centre on the hydrophobic character above and below the plane of amide bonds (personal communication with Oliver Korb). It may also explain why defectively packed backbone hydrogen bonds, which have been termed dehydrons, lead to hotspots at protein surfaces. [bib_ref] Dehydron: A Structurally Encoded Signal for Protein Interaction, Fernańdez [/bib_ref] At lower contour levels, the contributions from the methyl group are visible, again contributing unfavorably to the . At very low contour levels, one can observe the small and unfavorable contributions from water molecules in the second solvation shell . The presence of regions with high number density that contribute unfavorably to a favorable hydration free energy may seem counterintuitive. The reason why a vacuum is not present within such regions can be understood using a simple example. Consider water molecules in a voxel with weak solute−water interactions (E sw = −1.0 kcal/mol) and water−water interactions that are more favorable than in bulk (ΔE ww = −2.0 kcal/mol) that is strongly ordered (−TS sw = +3.0 kcal/ mol and −TΔS ww = +0.5 kcal/mol). For this voxel, ΔG IFST = +0.5 kcal/mol and the region contributes unfavorably to the solvation free energy. One could view these water molecules as "icelike" given their unfavorable entropy and favorable enthalpy compared with bulk water. However, if such a region is vacated (without allowing solvent rearrangement) then all surrounding voxels also lose their pair interactions and correlations with water molecules inside the voxel. The total change in free energy (going to this unphysical state) can thus be unfavorable. If subsequent solvent rearrangement around the vacuum region cannot overcome this loss, then the voxel will be occupied despite contributing unfavorably to the solvation free energy. Clearly, there are cases where water can rearrange around a vacuum, but water molecules at the vacuum interface are likely to show reduced water−water interactions. It is worth noting that the unfavorable contributions are smaller in magnitude than the favorable contributions. Considering all voxels in the case of acetamide, the most favorable relative free energy density is −0.28 kcal/mol/Å 3 whereas the least favorable relative free energy density is +0.04 kcal/mol/Å 3 . Because the exact free energy densities calculated will depend on the grid resolution and angular bin size used, it will be important to test this principle in future work. If these results accurately reflect the thermodynamics, an interesting consequence is the potential for hydration sites in protein binding sites that contribute unfavorably to the hydration free energy. Such sites would be major hotspots of binding. There is one additional issue which must be mentioned. ΔE IFST , −TΔS IFST , and ΔG IFST are calculated as the sum of contributions from each voxel inside a given volume V. The contributions from each voxel include pairwise contributions due to correlations with every other voxel. In the IFST framework, the pairwise contributions are split equally between the two voxels of the pair. Importantly, there are pairs where one of the voxels is not inside the volume V and thus there are regions outside the volume V which contribute to ΔE IFST , −TΔS IFST , and ΔG IFST . This must be considered when implementing IFST by counting pair contributions consistently. However, the contribution of voxels outside the volume V is expected to be very small if the volume V is significantly larger than the perturbing solute. In this case, a voxel outside V will only have significant correlations with voxels inside V that are close to the periodic boundary. These will have bulklike properties, leading to E sw ≈ 0, ΔE ww ≈ 0, S sw ≈ 0, ΔS ww ≈ 0, and ΔG IFST ≈ 0. # ■ conclusions IFST is a useful method to model solvation and has found particular application in understanding hydration phenomena in biological systems. [bib_ref] Thermodynamics of Buried Water Clusters at a Protein-Ligand Binding Interface, Li [/bib_ref] [bib_ref] Motifs for Molecular Recognition Exploiting Hydrophobic Enclosure in Protein-Ligand Binding, Young [/bib_ref] [bib_ref] High-Energy Water Sites Determine Peptide Binding Affinity and Specificity of PDZ Domains, Beuming [/bib_ref] [bib_ref] Thermodynamic Properties of Water Molecules at a Protein Protein Interaction Surface, Huggins [/bib_ref] However, there has been little analysis to assess the quantitative predictions of IFST. [bib_ref] Thermodynamics of Buried Water Clusters at a Protein-Ligand Binding Interface, Li [/bib_ref] This study addresses this issue by comparing hydration free energies from IFST with hydration free energies from FEP. The findings of this study are that IFST calculations show a good agreement with FEP calculations, with a mean unsigned difference of 0.7 kcal/mol in the predicted hydration free energies. Comparison of IFST predictions with the experimental hydration enthalpies and entropies suggests that there is a systematic overestimation of the entropies, which leads to a systematic underestimation of the free energies. For the CHARMM36 force field and the TIP4P-2005 water model, the agreement with the experimental hydration free energies is reasonable for FEP and IFST, with mean unsigned errors of 1.3 and 1.8 kcal/mol, respectively. Both IFST and FEP rely on the force field and are thus subject to the same problems in this respect. For IFST, the fine dependence of ΔE IFST on E bulk means that it is thus vital that the calculation of E bulk is converged correctly. E bulk will depend on the force field, water model, simulation package, and simulation parameters employed. Due to cancellation, this fine . Visualizations of the relative free energy density relative to bulk water for regions surrounding acetamide. Six views of acetamide, with the relative free energy density contoured at different levels. Favorable relative free energy density is contoured in red and unfavorable relative free energy density is contoured in yellow. The contouring for the favorable relative free energy increases from (A) to (B) to (C). The contouring for the unfavorable relative free energy decreases from (D) to (E) to (F). dependence will be of less importance in calculations of hydration free energy differences or binding energies, but only if the number of water molecules considered is similar. In addition, the systematic overestimations of −TΔS IFST are expected to be similar if the grid resolution and bin sizes are the same. Thus, predictions for different molecules and different regions are directly comparable for a given IFST implementation, but not necessarily between different IFST implementations. In addition to these results, there are a number of important points raised by the study. IFST has a number of limitations that should be addressed before widespread quantitative application. Current implementations are only rigorously valid for a fixed solute and this limits the utility. Flexible solutes can be considered in the IFST framework by performing an analysis on a number of solute conformations, 9 but this will require yet more data. One of the major approximations in IFST is the use of the KSAs for the water−water entropy calculations. Previous work on the solvation of methane suggests that these KSAs lead to reasonable results due to the cancellation of errors between more and less structured regions. However, this may not be the case for polar solutes and further testing of the KSAs should be a focus of future work. Sampling is also a problem for IFST. Previous work has explored the data requirements for convergence of the solvation energy and entropy. [bib_ref] Benchmarking the Thermodynamic Analysis of Water Molecules Around a Model Beta Sheet, Huggins [/bib_ref] [bib_ref] Application of Inhomogeneous Fluid Solvation Theory to Model the Distribution and Thermodynamics..., Huggins [/bib_ref] However, the comparison with FEP in this work allows a more thorough analysis. The results suggest that converged energies can be effectively sampled from 10 000 snapshots over a 20 ns simulation whereas the orientational component of the solute− water entropy term requires at least 5 000 000 snapshots over a 100 ns simulation to reach a converged prediction even with a modest angular bin size of 45°. However, the histogram method introduces a systematic overestimating bias to the entropies and this will scale with the volume of the system. Other work has shown that it is also difficult to estimate entropies using perturbation methods such as FEP and thermodynamic integration (TI). [bib_ref] Estimating Entropies from Molecular Dynamics Simulations, Peter [/bib_ref] In the context of IFST, assessment of the entropies may be more feasible using the k-NN algorithm, [bib_ref] Grid Inhomogeneous Solvation Theory: Hydration Structure and Thermodynamics of the Miniature Receptor..., Nguyen [/bib_ref] [bib_ref] Thermodynamic Properties of Liquid Aater: An Application of a Nonparametric Approach to..., Wang [/bib_ref] particularly for the increased sampling requirements of considering a highly flexible solute. It is difficult to fairly assess the computation times required, because the FEP calculations are integrated into NAMD whereas the IFST calculations are not. This requires postprocessing of largetrajectory files at considerable computational cost. Comparing just the simulation times, each solute was simulated for a total of 336 ns for FEP and 100 ns for IFST. While FEP is inherently more parallelizable than IFST in its current format, it should be possible to apply IFST to multiple independent MD simulations, which would parallelize it effectively. While suffering a number of drawbacks noted above, IFST does have a number of advantages over perturbation methods such as FEP and TI. Because IFST calculations are performed on a single simulation, there is no need for a lambda window schedule and no need to monitor overlap between lambda windows. In addition, there are no end points and thus there are no end-point catastrophes and no need for soft-core potentials. Importantly, there is no pathway and thus small and large perturbations can be considered with the same degree of sampling. However, perhaps the most useful feature of IFST is the spatial decomposition of the solvation free energies. This yields results that are more insightful than the total solvation free energies. Such visualization has also proved useful in 3D-RISM. [bib_ref] Spatial Decomposition of Solvation Free Energy Based on the 3D Integral Equation..., Yamazaki [/bib_ref] Indeed, it would be interesting to compare IFST results with the results of 3D-RISM 51 and cell theory, [bib_ref] Free Energy of Liquid Water from a Computer Simulation via Cell Theory, Henchman [/bib_ref] [bib_ref] Solvation Theory to Provide a Molecular Interpretation of the Hydrophobic Entropy Loss..., Irudayam [/bib_ref] which can provide spatially decomposed predictions of solvation free energies using different methods. This work represents a preliminary study on the quantitative application of IFST using a very small set of solutes. Future work should explore a number of avenues. A wide range of solutes would provide a sterner test for the methods. The inclusion of charged solutes may necessitate the use of a polarizable force field. Prior to this, it would be very useful to test the validity of the KSAs for a number of solutes, using the true inhomogeneous water−water correlation functions or via comparison with lower order entropy approximations. This should be done in concert with a thorough determination of the time scales and sampling requirements that are necessary for effective convergence of the thermodynamic predictions at the desired level of accuracy. In addition, care must be taken to employ methods that effectively capture the underlying probability distributions. The combination of ΔG IFST with an assessment of protein−ligand interactions will facilitate the calculation of binding free energies. This will augment binding affinity predictions based on a single unbound state simulation, 54 albeit at increased computational cost. To achieve this, it will be necessary to incorporate solute flexibility into IFST. [bib_ref] Grid Inhomogeneous Solvation Theory: Hydration Structure and Thermodynamics of the Miniature Receptor..., Nguyen [/bib_ref] In conclusion, the agreement between hydration free energy predictions for IFST and FEP is very encouraging. It is clear that the entropic contributions are overestimated by IFST and further work is needed. However, the insight gained by visualizing the relative free energy densities provides a major advantage of IFST that make this a worthwhile objective. ## ■ author information Corresponding Author *E-mail: [email protected]. Tel.: +44(0)1223763367. # Author contributions The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. [fig] Figure 1: Convergence of E bulk for a 100 ns simulation of bulk TIP4P-2005 water: the running average of E bulk for a 100 ns NPT simulation of bulk TIP4P-2005 water at 300 K and 1 atm. The running average is calculated for 400 blocks of 250 ps. Each block is the average potential energy per water molecule taken from 2500 samples with one every 100 fs. [/fig] [fig] Figure 2: Translational correlation function for TIP4P-2005 between 0.0 and 3.6 Å. [/fig] [fig] Figure 3: Cumulative orientational contribution to TS bulk for bulk TIP4P-2005 water: the cumulative contribution to TS bulk between 0.0 and 3.6 Å from the bulk water simulation of TIP4P-2005 using the radial coordinate system (blue line) and the Cartesian coordinate system (red line). [/fig] [fig] Figure 4: Cumulative contributions to ΔE IFST , ΔS IFST , and ΔG IFST for acetamide at increasing distances from the origin: the cumulative contributions to ΔE IFST (blue diamonds), TΔS IFST (red squares), and ΔG IFST (green triangles) for acetamide between 0.0 and 12.0 Å from the origin. [/fig] [fig] Figure 5: Correlation plots between ΔG, ΔH, and TΔS for experiment and the predictions of FEP and IFST. Plots of the correlation between (a) ΔG IFST and ΔG FEP with blue diamonds, (b) ΔG IFST and ΔG Experimental with blue triangles, (c) ΔH IFST and ΔH Experimental with red triangles, and (d) TΔS IFST and TΔS Experimental with green triangles. [/fig] [table] Table 1: Lambda Schedules for FEP Annihilation and Creation a The 24 FEP windows used in the annihilation and creation simulations of the six solutes. The initial and final values of λ are given for each window. [/table] [table] Table 2: Results of IFST Calculations on Bulk Water and the Six Solutes a The calculated IFST quantities for bulk water and the six solutes from the 100 ns simulations. The total translational and orientational contributions are the sums of the solute−water and water−water terms. [/table] [table] Table 3: Convergence of IFST Predictions with IncreasingSimulation Time a [/table] [table] Table 5: Effect of the Grid Resolution on the Translational Component of −TS sw [/table] [table] Table 6: Effect of the Angular Bin Size on the Orientational Component of −TS sw [/table] [table] Table 7: Results of the FEP and IFST Calculations Compared to Experiment a [/table]

Increased HIV Prevention Program Coverage and Decline in HIV Prevalence Among Female Sex Workers in South India Background: As one way of assessing the impact of Avahan, the India AIDS Initiative of the Bill & Melinda Gates Foundation, we examined the association between HIV prevention program indicators and changes in HIV prevalence among female sex workers (FSWs) between 2005 and 2009. Methods: We conducted a secondary data analysis from 2 large crosssectional surveys (2005Y2006 and 2008Y2009) across 24 districts in south India (n = 11,000 per round). A random-effect multilevel logistic regression analysis was performed using HIV as the outcome, with individual independent variables (from both surveys) at level 1 and district-level FSW-specific program indicators and contextual variables at level 2. Program indicators included their 2006 value, the difference in their values between 2008 and 2006, and the interaction between this difference and study round.Results: HIV prevalence among FSWs decreased from 17.0% to 14.2% (P G 0.001). This decline varied significantly (P = 0.006) across levels of difference in program coverage (% of FSWs contacted by the program in a given year). Odds ratios comparing HIV prevalence between rounds changed with the level of increase in coverage and were statistically significant with coverage increase Q quartile (Q) 1: odds ratio, 0.85 at Q1; 0.78 at Q2; 0.66 at Q3; and 0.51 at Q4.Conclusions: These findings suggest that increased program coverage was associated with declining HIV prevalence among FSWs covered by the Avahan program. The triangulation of our results with those from other approaches used in evaluating Avahan suggests a major impact of this intervention on the HIV epidemic in southern India. ## A vahan, the india aids initiative of the bill & melinda gates Foundation, is a comprehensive HIV prevention program targeting the most at-risk populations in the states of India most affected by the HIV epidemic (Andhra Pradesh, Karnataka, Maharashtra, and Tamil Nadu in the south of the country, and Manipur and Nagaland in the northeast). It was initially implemented through a network of nongovernmental organizations (NGOs) and has progressively been transferred to government agencies and community-based organizations since 2008. Because cluster randomized designs have major limitations for evaluating complex large-scale interventions, 1,2 the initial impact evaluation plan for Avahan included serial cross-sectional studies in the high-risk populations, based on which sophisticated mathematical models were developed to estimate program impact in those populations, and in the general population. [bib_ref] Evaluation design for large-scale HIV prevention programmes: The case of Avahan, the..., Chandrasekaran [/bib_ref] [bib_ref] Evaluating large-scale HIV prevention interventions: Study design for an integrated mathematical modelling..., Boily [/bib_ref] The use of this modeling approach has been complicated by the lack of adequate baseline data, 4 but recently, published results using this approach suggest a major impact of Avahan on the HIV epidemic in [bib_ref] Interim modelling analysis to validate reported increases in condom use and assess..., Pickles [/bib_ref] and in the 4 South India states mentioned above. [bib_ref] Assessment of the population-level effectiveness of the Avahan HIV-prevention programme in South..., Pickles [/bib_ref] A purely statistical approach based on HIV surveillance data among pregnant women was also used to estimate the overall Avahan impact in the 6 Avahan states. [bib_ref] Assessment of populationlevel effect of Avahan, an HIV-prevention initiative in India, Ng [/bib_ref] Although suggesting a positive impact of the intervention in the general population, this study has the limitation of not taking into account the nature of the Avahan intervention, including its various components, and does not allow for the analysis of the actual mechanism leading to the impact. [bib_ref] Beyond accountability: Learning from largescale evaluations, Boerma [/bib_ref] We examined changes over time occurring in HIV prevalence among female sex workers (FSWs), the largest risk population in the program, between 2 large surveys carried out in 2005 to 2006 and 2008 to 2009, in 24 districts of the 4 southern states of India covered by Avahan, where the HIV epidemic is predominantly heterosexual and mainly driven by sex work. [bib_ref] Ecological analysis of the association between high-risk population parameters and HIV prevalence..., Alary [/bib_ref] [bib_ref] To what extent is the HIV epidemic in southern India driven by..., Vickerman [/bib_ref] As indicators related to program coverage and implementation have shown very sharp increases between baseline and 2008, [bib_ref] Scale-up and coverage of Avahan: A large-scale HIV-prevention programme among female sex..., Verma [/bib_ref] we considered the impact of these increases on the changes in HIV prevalence between surveys. Given the heterogeneity of the HIV epidemic in India, [bib_ref] Heterogeneity of the HIV epidemic in the general population of Karnataka state,..., Banandur [/bib_ref] it is important to also consider the influence of broader contextual factors that may have an impact on the HIV epidemic in high-risk groups. We therefore carried out a multilevel statistical analysis of the individual, contextual and program factors impacting on HIV prevalence among FSWs, and on the changes in the prevalence levels between the 2 survey rounds. # Methods data sources The design and study procedures of the cross-sectional studies, known as integrated behavioral and biological assessments (IBBAs), have been described in detail elsewhere [bib_ref] Determinants of HIV prevalence among female sex workers in four south Indian..., Ramesh [/bib_ref] [bib_ref] Baseline integrated behavioural and biological assessment among most at-risk populations in six..., Saidel [/bib_ref] and are summarized in the Supplementary Online Material Methods section, http://links.lww.com/OLQ/A86, as are the main components of the Avahan intervention. The common set of program indicators for all districts covered by Avahan has been described extensively elsewhere. [bib_ref] Scale-up and coverage of Avahan: A large-scale HIV-prevention programme among female sex..., Verma [/bib_ref] For the purpose of the present analysis, we used data collected by the NGOs implementing the intervention in each district along with complementary information to estimate: (1) program coverage (proportion of FSWs contacted by the program at least once in a given year), (2) the proportion of FSWs contacted monthly, and (3) We considered a total of 53 district-level contextual variables for inclusion in the multilevel statistical model (see complete list in the Supplementary Online Material http://links.lww.com/OLQ/A87). The study and its consent procedures were approved by the ethics committees of all institutions that were involved in the data collection for this study (see Supplementary Online Material Methods section for more details on ethical considerations http://links.lww.com/OLQ/A86). ## Statistical analyses We assessed differences in personal characteristics between participants who were tested for HIV and those who were not, and between participants in round 1 and those in round 2, using the W 2 test. We compared HIV prevalence between the study rounds for each of the 24 IBBA districts. We then built multilevel (FSW personal characteristics at level 1 and districtlevel contextual and program variables at level 2) logistic regression models with HIV prevalence as the outcome, using the round 1 and round 2 merged data set. Study round was considered as the main independent variable in our analyses because the difference in HIV prevalence between the rounds was our central interest. We also preselected a certain number of the individual variables from the questionnaire for inclusion as independent variables in the model because they could be confounders of the differences observed between rounds. This was done based on variables previously identified to be associated with HIV in the first IBBA round [bib_ref] Determinants of HIV prevalence among female sex workers in four south Indian..., Ramesh [/bib_ref] and of a priori knowledge from the literature (see the Supplementary Online . We entered all of these variables into a multilevel logistic regression model with a random intercept for the districts and a random coefficient for round because the difference in HIV prevalence varied widely between districts. We then removed sequentially from this model all the individual variables with P 9 0.05, as long as they were not confounding the other associations, to arrive at a model with individual variables only. We preselected the contextual variables for addition to the models above based on linear regression models with the aggregate HIV prevalence rate in each district as the dependent variable and the contextual variables as the independent variables. Those with a P value less than 0.05 in these models were then added to the previous multilevel models. Baseline HIV prevalence among FSWs in each district was also included in the model as an additional district-specific variable. The removal of nonsignificant variables at this stage led to the basic model with individual and district-level contextual variables. To the basic model, we then added the 3 pairs of districtlevel program variables (2006 value and difference between 2008 and 2006), along with the interaction terms between the 2008 to 2006 difference and the round. We kept in the model only the program variables corresponding to a significant interaction (P G 0.05) with the round. Finally, because we were interested in factors that could impact the differences in the outcomes observed between the IBBA rounds, we examined oneby-one the interaction terms between the round and each of the other individual and district-level contextual factors included in the first multilevel model described above. We kept the statistical interactions that reached P G 0.05 in these models. This led to the final model, with assessment of the intervention impact, through the interaction terms between the study round and the 2008 to 2006 differences in program variables, and inclusion of other factors having a multiplicative statistical interaction with the round. All analyses described above were carried out using STATA IC 11.1 for Windows (StataCorp LP, College Station, TX). # Results Among the 14,601 women approached in round 1, 11,690 (80.1%) answered the questionnaire. Among the latter, 10,806 were tested for HIV (92.4%), 10,716 (91.7%) for syphilis, and 10,674 (91.3%) for gonorrhea and chlamydia (NG/CT). The overall participation rate was slightly lower in 2008: 15,682 women were approached, of whom 11,486 (73.2%) answered the questionnaire; however, among the participants, 10,901 (95.0%) were tested for HIV, 10,806 (94.1%) for syphilis and 10,880 (94.8%) for NG/CT. No data were available to compare participants with nonparticipants. However, [fig_ref] TABLE 1: Comparison of the Characteristics of Female Sex Workers Providing and Not Providing... [/fig_ref] shows the comparison between participants who were tested for HIV and those who were interviewed only. There were significant differences for most variables examined. This is, however, largely due to the very large sample sizes of these surveys. In addition, the proportions among all participants were almost identical to those among those tested for HIV (see the ''Total'' columns in [fig_ref] TABLE 1: Comparison of the Characteristics of Female Sex Workers Providing and Not Providing... [/fig_ref]. [fig_ref] TABLE 2: Comparison of Selected Individual Characteristics of Female Sex Workers Between Survey Rounds... [/fig_ref] shows the comparison between various characteristics of participating FSWs between rounds 1 and 2. The differences were significant for most variables examined. However, this was mostly for behavior or infections that were targeted by the Avahan program, such as condom use, violence, NG/CT, and syphilis prevalence. For the other significant variables, the actual differences in the distribution of their values between rounds were mostly small, with the statistical significance often due to the very large sample sizes of these surveys. HIV prevalence declined significantly from 17.0% to 14.2% (P G 0.001) from 2005Y2006 to 2008Y2009 [fig_ref] TABLE 3: Comparison of HIV Prevalence [/fig_ref]. This decline was significant in 2 of the 4 states and 10 of the 24 districts. There was an increase in HIV prevalence in 4 districts, but it was statistically significant in only 1. [fig_ref] TABLE 3: Comparison of HIV Prevalence [/fig_ref] also shows the variation in program coverage and condom requirement met between the study rounds. There was a significant correlation between changes in HIV prevalence between 2006 and 2008 and changes in coverage (Spearman r = j0.410, P = 0.048), whereas such an association was borderline significant when considering condom requirement met (Spearman r = j0.398, P = 0.054). ## Decline in hiv prevalence in fswsvindia Sexually Transmitted Diseases & Volume 41, Number 6, June 2014 The 2 latter program indicators were also highly correlated together (Spearman r = 0.647, P = 0.006). The difference in the proportion of FSWs contacted on a monthly basis between 2006 and 2008 was not significantly correlated with the changes in HIV prevalence (Spearman correlation coefficient = j0.1992, P = 0.351) or with the changes in the other program indicators (data not shown). The left columns of [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref] show the results of the multilevel model without program variables and interaction terms. The adjusted odds ratio (AOR) comparing HIV prevalence in round 2 with that in round 1 was 0.76, with a narrow 95% confidence interval (95% CI) of 0.67 to 0.85. A number of demographic and behavioral variables were associated with HIV [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref]. Surprisingly, women reporting consistent condom use with clients and carrying condoms with them had a significantly higher HIV prevalence. The only significant contextual variable among those examined was mean age at marriage of girls, with an approximately 8% lower HIV prevalence for each year of increase in this district-level variable. As expected, baseline HIV prevalence was also strongly associated with overall HIV prevalence. The random coefficient for the round had a variance significantly higher than zero, reflecting the high level of heterogeneity in the changes in HIV prevalence across districts. The variance of the random intercept, although much lower than in the null model, was also significantly different from zero [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref]. The right columns of [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref] show that there was a significant negative interaction between the round and changes in program coverage between 2006 and 2008. The interaction terms between the round and changes in other program variables were not statistically significant and thus not kept in the model. Both baseline HIV prevalence and having a regular nonpaying partner also had significant multiplicative interactions with survey round. The only variable not involved in the interaction terms that did not remain significant compared with the previous model was mean age at marriage of girls at the district level. The variance of the random coefficient for the round, though significantly different from zero, was lower than that in the previous model, whereas the variance of the random intercept was now close to null in this model [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref]. [fig_ref] TABLE 5: Changes Observed in HIV Prevalence Among FSWs Between the Study Rounds [/fig_ref] shows the decreases in HIV prevalence for the various categories of variables shown to have an interaction with the study round from the results presented in [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref]. The decrease in HIV prevalence was not as strong among women with a main nonpaying partner compared with those without, although the latter had a higher overall HIV prevalence. The decrease in HIV prevalence was not significant in districts with very low baseline HIV prevalence and significant for higher values starting at about the first quartile of this variable. Finally, there was a stronger decline in HIV prevalence in districts with a larger increase in program coverage between 2006 and 2008 [fig_ref] TABLE 5: Changes Observed in HIV Prevalence Among FSWs Between the Study Rounds [/fig_ref]. The decrease in HIV prevalence was not significant when coverage was higher in 2006 than in 2008. This analysis controlled for a number of individual and contextual factors that could have potentially confounded this association, as well as for baseline HIV prevalence at the district level and its interaction with the round, thus taking into account the fact that decreases in prevalence were more likely to occur in districts with higher baseline values. The use of a random coefficient for the round took into account the wide variability in the changes in HIV prevalence between districts. # Discussion It was difficult to fully address the impact of the program indicator on condom requirement met because of the high correlation between its 2006 to 2008 district-level variation and that of program coverage. However, the borderline significant correlation between increases in condom requirement met and decreases in HIV prevalence at the district-level suggests that this indicator is also part of the mechanism leading to impact. Finally, it is possible that the actual coverage of the program is more important in terms of impact than the frequency of the contacts with the target population, as long as some (unknown) threshold is reached for the latter. The associations that we found between HIV prevalence and the individual variables considered in the analysis are largely similar to what was found in an analysis of round 1 data. 14 That condom use and condom carriage were both associated with a higher HIV prevalence may be due to reverse causation, with FSWs having acquired HIV before being reached by the interventions and starting to use condoms once they learned more about HIV, especially when they discovered their serological status. A similar phenomenon was observed in studies of the general population in southern India. 13,16 Among the contextual district-level variables considered, only mean age at marriage of girls was significantly associated with HIV in the basic model. Interestingly, this factor was also associated with HIV prevalence among pregnant women attending antenatal care clinics in the same IBBA districts. [bib_ref] Multi-level analysis of the predictors of HIV prevalence among pregnant women enrolled..., Thamattoor [/bib_ref] However, in the model with program variables, mean age at marriage was not a significant predictor of HIV. Careful examination of the data showed that it was the introduction of the interaction between baseline HIV prevalence and the round that was mainly confounding this association (data not shown). The results of this study constitute a significant addition to the overall picture of the Avahan impact evaluation. Whereas it has been shown that the implementation of the program was highly successful, 12 that favorable time trends in intermediate outcomes (e.g., condom use and prevalence of curable sexually transmitted infections) occurred after implementation, 18Y20 that condom use was positively associated 21 and sexually transmitted infection prevalence negatively associated [bib_ref] Role of community group exposure in reducing sexually transmitted infectionYrelated risk among..., Yadav [/bib_ref] with exposure to the intervention, and that the program seemingly had an impact on HIV prevalence at the general population level, [bib_ref] Assessment of populationlevel effect of Avahan, an HIV-prevention initiative in India, Ng [/bib_ref] [bib_ref] Population-level impact of Avahan in Karnataka state, south India using multilevel statistical..., Banandur [/bib_ref] the evidence for an impact on HIV prevalence among FSWs has so far been limited to time trends observed [bib_ref] Changes in risk behaviours and prevalence of sexually transmitted infections following HIV..., Ramesh [/bib_ref] [bib_ref] Declines in risk behaviour and sexually transmitted infection prevalence following a communityled..., Beattie [/bib_ref] and mathematical modeling studies 5Y7 based on these trends. This is the first study showing a direct relationship between program exposure assessed through routine program monitoring and decrease in HIV prevalence in one of the vulnerable populations targeted by the intervention. However, there are a number of limitations to this study. First, there was no randomization of the intervention and the assessment of the impact relies only on serial observational data. The decision not to use a randomized design for this evaluation was taken right at the start for reasons that have been summarized in previous publications. [bib_ref] Evaluation design for large-scale HIV prevention programmes: The case of Avahan, the..., Chandrasekaran [/bib_ref] [bib_ref] Evaluating large-scale HIV prevention interventions: Study design for an integrated mathematical modelling..., Boily [/bib_ref] The limitations of communityrandomized trials for ''real-world'' evaluations have since that time been acknowledged more widely. [bib_ref] Evaluating HIV prevention effectiveness: The perfect as the enemy of the good, Laga [/bib_ref] Furthermore, we were able to demonstrate an association between the level of intervention coverage and decreases in HIV prevalence at the district level, thus reaching a level of plausibility in the attribution of the impact on HIV prevalence to Avahan as per the framework proposed by Habicht et al. [bib_ref] Evaluation designs for adequacy, plausibility and probability of public health programme performance..., Habicht [/bib_ref] Second, we do not know the characteristics of the women who declined participation in the surveys and the proportion of refusals increased between the 2 rounds. It is impossible to assess the impact of refusals on the observed trends because no information is available on the women who did not participate. In addition, there were significant differences between women who provided biological samples and those who only accepted to be interviewed. However, for most variables, these differences were of small magnitude, given the very large sample sizes, or went in the same direction in both survey rounds. Moreover, the distribution of the variables considered in [fig_ref] TABLE 1: Comparison of the Characteristics of Female Sex Workers Providing and Not Providing... [/fig_ref] among all participants was almost identical to that among those tested for HIV, because the proportion of FSWs not providing a sample among the participants was low in both rounds. Thus, important biases resulting from these differences when comparing HIV prevalence in both rounds are unlikely. Similar observations are relevant to the comparison of the characteristics of the participants between the rounds [fig_ref] TABLE 2: Comparison of Selected Individual Characteristics of Female Sex Workers Between Survey Rounds... [/fig_ref] : apart for the changes expected because of the intervention, the absolute differences were mostly small, although often statistically significant because of the very large sample sizes. [bib_ref] Dramatic increases in HIV prevalence after scale-up of antiretroviral treatment: A longitudinal..., Zaidi [/bib_ref] Consequently, if the increase in ART coverage between the study rounds had any impact on HIV prevalence trends during this period, it would likely have been to artificially mask a downward trend. Finally, the first survey was carried out after more than 1 year of intervention in most districts, and the level of intervention coverage had already reached relatively high levels at that time; this could have led to an underestimation of the decrease of HIV prevalence between the study rounds and of the association between increases in program indicators and declines in HIV prevalence. In conclusion, this study strongly suggests that the Avahan intervention, where appropriately implemented, had a favorable impact on HIV prevalence among FSWs in the 4 southern states [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref] because here, for the sake of simplicity, we used 3 different models with only 1 interaction variable in each of them (whereas in [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref] , we used 1 model including all the interaction terms); however, the AOR corresponding to each of the main variable and its interaction with round was similar in the 3 separate models and the model used for [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref]. † AOR comparing HIV prevalence in round 2 with that in round 1 at different levels of each independent variable interacting with the round (adjusted for all the independent variables listed in [fig_ref] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level... [/fig_ref]. ‡ 95% CI of the AOR. § At the district level. ¶ Coverage can be more than 100% because the denominator of this indicator is based on the estimate of the size of the FSW population at any moment in the district, whereas the number of women actually covered in the district could be higher than the denominator in places with high turnover of FSWs (mean duration of presence of the FSWs in the district of less than 1 year). of India covered by the intervention. The triangulation of our results with those from other approaches used in the Avahan evaluation 5Y8,23 suggests a major overall impact of this program on the HIV epidemic in southern India. Interventions targeting most at-risk populations, such as FSWs, should be scaled up in all countries where specific vulnerable populations contribute significantly to the spread of HIV. [fig] *: Percentage of the FSW population contacted at least once by the program in the previous year. This was computed from the number of FSWs contacted at least once by the program in the previous year divided by the estimation of the size of the FSW population on the same year. † Percentage of the condoms required by FSWs for all sex acts with clients in a given year. This was computed based on the number of condoms distributed to FSWs by the NGOs in a given year as the numerator, whereas the denominator was the expected number of condoms needed by FSWs in the district, based on the estimated number of FSWs in the district and the mean number of client-contacts estimated from the cross-sectional survey data. [/fig] [table] TABLE 1: Comparison of the Characteristics of Female Sex Workers Providing and Not Providing a Biological Sample Among ThoseInterviewed in Survey Rounds 1 and 2 *P value for the W 2 test comparing the distribution of characteristics of participants providing and those not providing a biological sample, with appropriate degrees of freedom [/table] [table] TABLE 2: Comparison of Selected Individual Characteristics of Female Sex Workers Between Survey Rounds 1 and 2 *P value for the W 2 test comparing the distribution of characteristics of participants between the study rounds. † Infection by either Neisseria gonorrhoeae or Chlamydia trachomatis. [/table] [table] TABLE 3: Comparison of HIV Prevalence (in percent), Program Coverage* and Condom Requirement Met † Between 2 Cross-Sectional Surveys Carried Out in 2005 to 2006 (Round 1) and 2008 to 2009 (Round 2) Among FSWs in 24 Districts of Four Southern States of India [/table] [table] TABLE 4: Results of the Multilevel Logistic Regression Models of Individual-, Programmatic-, and District-Level Determinants of HIV Prevalence Among FSWs in 24 Districts in South India, 2005 to 2009 Decline in HIV Prevalence in FSWsVIndia Sexually Transmitted Diseases & Volume 41, Number 6, June 2014 Third, given the potential of antiretroviral therapy (ART) to decrease HIV transmission, 25 the increase in ART coverage between the study rounds could have been involved in the decrease in HIV prevalence we observed, whereas it could not been directly assessed because information on ART use by HIV-infected women was not collected in the IBBA surveys. This favorable impact of ART on HIV prevalence is, however, unlikely in our study. Indeed, out of an estimated 2.6 million HIV-infected people in India, only 6845 received ART in 2005, whereas coverage increased to 140,654 people on treatment (approximately 5% of all infected people) in 2008 and to 223,223 (approximately 9% of all infected people) in 2009. 26 Antiretroviral therapy coverage thus remained relatively low until 2008 to 2009, by which time IBBA surveys among FSWs were completed in all districts. In addition, as ART improves survival, its expansion could have first led to an increase in HIV prevalence, despite a reduction in new HIV cases, as was recently observed in a highly affected community in South Africa. [/table] [table] TABLE 5: Changes Observed in HIV Prevalence Among FSWs Between the Study Rounds (2005Y2006 and 2008Y2009) at Different Levels of the Independent Variables Having a Significant Statistical Interaction With the Study Round*, 24 Districts of South India *The results presented in this table differ slightly from those in [/table]

Influence of ECAP process on mechanical and corrosion properties of pure Mg and ZK60 magnesium alloy for biodegradable stent applications # Introduction Nowadays, biomaterial technology is built on traditional permanent metallic materials including stainless steel, Nitinol and cobalt-chromium alloys. In particular, these inert materials are used as load-bearing implants for replacement of diseased or damaged tissues. However, some particular disadvantages, including physical irritation, chronic inflammatory inconvenience, longterm endothelial dysfunction and, more importantly in pediatric and adolescent patients, a second intervention is required for implant removal, causing adverse tissue reactions. [bib_ref] Adverse Tissue Reactions to Bioabsorbable Fixation Devices, Böstman [/bib_ref] [bib_ref] The road to bioabsorbable stents: reaching clinical reality?, Erne [/bib_ref] [bib_ref] Biodegradable metals for cardiovascular stent application: interests and new opportunities, Moravej [/bib_ref] More recently, biodegradable materials like magnesium and iron alloys have been proposed in medical science as a novel class of highly bioactive materials. That is, these materials are supposed to temporarily aid the healing process of a diseased tissue or organ and then they can progressively disappear by virtue of body fluids corrosion after a certain length of functional use, leading to simultaneous implant replacement through the surrounding tissue. [bib_ref] Degradable biomaterials based on magnesium corrosion, Witte [/bib_ref] [bib_ref] Developments in metallic biodegradable stents, Hermawan [/bib_ref] [bib_ref] In vivo corrosion of four magnesium alloys and the associated bone response, Witte [/bib_ref] Magnesium is one of the most promising candidates for biodegradable applications due to its biocompatibility as an essential element to human metabolism. Several studies have proven that the daily intake of Mg for a normal adult exceeds 300 mg and excessive magnesium cations possibly produces by degradation could be safely excreted in the urine. [bib_ref] In vivo corrosion of four magnesium alloys and the associated bone response, Witte [/bib_ref] [bib_ref] In vitro corrosion and biocompatibility of binary magnesium alloys, Gu [/bib_ref] [bib_ref] Research on an Mg-Zn alloy as a degradable biomaterial, Zhang [/bib_ref] [bib_ref] Safety and efficacy of bioabsorbable magnesium alloy stents in porcine coronary arteries, Waksman [/bib_ref] [bib_ref] PROGRESS-AMS (Clinical Performance and Angiographic Results of Coronary Stenting with Absorbable Metal..., Erbel [/bib_ref] [bib_ref] Magnesium: nutrition and metabolism, Vormann [/bib_ref] However, the greatest limitation of Mg consists of its fast corrosion rate, especially in human body fluid containing chloride, giving rise to immature drop in mechanical integrity of the device before accomplishing its defined mission. [bib_ref] Understanding Magnesium Corrosion-A Framework for Improved Alloy Performance, Song [/bib_ref] [bib_ref] Control of biodegradation of biocompatable magnesium alloys, Song [/bib_ref] Over and above corrosion rate, critical hydrogen gas bubbles and alkalization resulting from corrosion of Mg in body fluid are also other problematic subjects in fast corrosion-rate processes, leading to accumulation of evolved hydrogen bubbles in gas pockets next to the implant and consequently, causing necrosis of tissues. However, if the corrosion rate of Mg implant can be suitably controlled, hydrogen evolution will not be rapid enough to cause critical subcutaneous bubbles, and the alkalization effect could be easily balanced by metabolic mechanisms in the human body. [bib_ref] A Possible Biodegradable Magnesium Implant Material, Song [/bib_ref] [bib_ref] Recent Progress in Corrosion and Protection of Magnesium Alloys, Song [/bib_ref] [bib_ref] Control of Degradation of Biocompatible Magnesium in a Pseudo-Physiological Environment by a..., Song [/bib_ref] equal channel angular pressing (eCAp) was performed on ZK60 alloy and pure Mg in the temperature range 150-250 °C. A significant grain refinement was detected after eCAp, leading to an ultrafine grain size (UFG) and enhanced formability during extrusion process. Comparing to conventional coarse grained samples, fracture elongation of pure Mg and ZK60 alloy were significantly improved by 130% and 100%, respectively, while the tensile strength remained at high level. extrusion was performed on eCAp processed billets to produce small tubes (with outer/inner diameter of 4/2.5 mm) as precursors for biodegradable stents. Studies on extruded tubes revealed that even after extrusion the microstructure and microhardness of the UFG ZK60 alloy were almost stable. Furthermore, pure Mg tubes showed an additional improvement in terms of grain refining and mechanical properties after extrusion. electrochemical analyses and microstructural assessments after corrosion tests demonstrated two major influential factors in corrosion behavior of the investigated materials. the presence of Zn and Zr as alloying elements simultaneously increases the nobility by formation of a protective film and increase the local corrosion damage by amplifying the pitting development. eCAp treatment decreases the size of the second phase particles thus improving microstructure homogeneity, thereby decreasing the localized corrosion effects. A further challenging issue to be faced concerns mechanical properties. Generally, Mg alloys have lower mechanical properties than commonly used inert biomaterials. Considering stent applications, fracture of the stent strut during expansion also has to be avoided by enhancing alloy ductility. Therefore, controlling the corrosion rate to achieve a prolonged and uniform degradation of the device made by alloys with reasonable strength and ductility are key factors in the development of Mg implants and stents. [bib_ref] Biodegradable metals for cardiovascular stent application: interests and new opportunities, Moravej [/bib_ref] [bib_ref] Control of biodegradation of biocompatable magnesium alloys, Song [/bib_ref] [bib_ref] Benestent Study Group. A comparison of balloon-expandable-stent implantation with balloon angioplasty in..., Serruys [/bib_ref] In general, the most efficient approach to improve both mechanical properties and corrosion resistance of magnesium is adding specific alloying elements. There are two basic groups of Mg alloys namely those containing aluminum and the class of aluminum free alloys. 4,6-8 However, it has been reported that for the use in humans, aluminum free alloy systems are recommended since aluminum ions can combine with inorganic phosphates, causing a lack of phosphate in the human body. [bib_ref] Degradable biomaterials based on magnesium corrosion, Witte [/bib_ref] On the other hand, it is cited that addition of rare earth (RE) elements (Ce, Y, etc.) could lead to hepatotoxicity and also excessive yttrium ions induces adverse effects on DNA transcription factors. [bib_ref] Differences in behavior among the chlorides of seven rare earth elements administered..., Nakamura [/bib_ref] [bib_ref] Effect of Long-Term Intake of Y3+ in Drinking Water on Gene Expression..., Yang [/bib_ref] It is therefore recognized that alloying elements must be carefully selected in order to meet the biocompatibility requirements. [bib_ref] In vitro corrosion and biocompatibility of binary magnesium alloys, Gu [/bib_ref] [bib_ref] A Possible Biodegradable Magnesium Implant Material, Song [/bib_ref] Since it is demonstrated that Zn is an essential nutritive element and its ions can be absorbed without harming vital organs, this element could be the first choice among alloying elements. Zirconium, particularly in small amount, less that 0.8 wt%, is considered as a biocompatible element. [bib_ref] Degradable biomaterials based on magnesium corrosion, Witte [/bib_ref] [bib_ref] Grain refinement of magnesium alloys, Stjohn [/bib_ref] [bib_ref] Histological response to cylinders of a low modulus titanium alloy (Ti-13Nb-13Zr) and..., Goodman [/bib_ref] Accordingly, it is proven that introducing of Zn or Zr to pure Mg brought about improved mechanical properties and corrosion resistance as well as good cytocompatibility. [bib_ref] In vitro corrosion and biocompatibility of binary magnesium alloys, Gu [/bib_ref] [bib_ref] Investigation of the corrosion for Mg-xGd-3Y-0.4Zr (x = 6, 8, 10, 12..., Chang [/bib_ref] [bib_ref] The relation between microstructure and corrosion behavior of Mg-Y-RE-Zr alloys, Ben-Hamu [/bib_ref] In addition, several studies reported that grain refinement induced by processing techniques based on severe plastic deformation (SPD) is an effective approach to improve both mechanical and corrosion properties of Mg alloys due to Hall-Petch strengthening and homogeneous distribution of precipitates, respectively. [bib_ref] Enhanced corrosion resistance of Mg alloy ZK60 after processing by integrated extrusion..., Orlov [/bib_ref] [bib_ref] Effect of grain size and twins on corrosion behaviour of AZ31B magnesium..., Aung [/bib_ref] [bib_ref] Principles of equal-channel angular pressing as a processing tool for grain refinement, Valiev [/bib_ref] [bib_ref] The processing of ultrafine-grained Mg tubes for biodegradable stents, Ge [/bib_ref] In recent years, as a novel metal forming process, Equal-channel angular pressing (ECAP) has been widely investigated on Mg alloys. However, those attempts generally cover works on mechanical properties assessments 28,31-33 and texture monitoring. [bib_ref] Orientation stability in equal channel angular extrusion. Part II: Hexagonal close-packed materials, Li [/bib_ref] [bib_ref] crystallographic texture and mechanical properties of the magnesium alloy AZ31B after different..., Seipp [/bib_ref] A couple of works have also investigated typical biomedical application based behavior of ECAPed Mg alloys. [bib_ref] Safety and efficacy of bioabsorbable magnesium alloy stents in porcine coronary arteries, Waksman [/bib_ref] [bib_ref] PROGRESS-AMS (Clinical Performance and Angiographic Results of Coronary Stenting with Absorbable Metal..., Erbel [/bib_ref] [bib_ref] Magnesium: nutrition and metabolism, Vormann [/bib_ref] [bib_ref] Understanding Magnesium Corrosion-A Framework for Improved Alloy Performance, Song [/bib_ref] [bib_ref] Control of biodegradation of biocompatable magnesium alloys, Song [/bib_ref] Accordingly, a vacancy is felt about the bio-application based exploration of pure Mg, that is tried to be addressed in this work. Moreover, to the best of authors' knowledge, no systematic investigation of ECAP treated Mg based materials for stents application has been reported in the literature. Efforts have been put on elucidation of the primary steps of this area in this work. Another objective of this work has been spotlighting the potential of ECAP process for supplying precursor materials for subsequent forming steps, such as extrusion, while keeping the favorable properties accessed by the very ECAP step, namely, ultrafined grain (UFG) structure. Interestingly, ECAP induced UFG structure, which has been an area of intense research and interest due to its excellent mechanical specifications, was retained even after ulterior treatments such as extrusion. In practice, this can be translated to a geometry close to final application (cylinder for stent in this case) with outstanding mechanical properties of a UFG structure. Further electrochemical measurements were also conducted to provide some basic information about the effect of such mechanical treatment on the corrosion resistance of the mater. # Results and discussion ## Microstructure The microstructure of the as-received billets is shown in [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref]. the MA (see the experimental part for reference to samples naming) sample possessed a quite coarse grain structure with average grain size of about 250 µm, whereas the AA sample featured a relatively finer and heterogeneous microstructure, with a bimodal grain size distribution made up of coarse grains of approximately 10-15 µm in size together with very fine grains of about 2-3µm [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref]. In order to induce grain refinement in MA and AA billets without any cracking during ECAP, initial processing temperature had to be increased up to 275 °C and 250 °C for MA and AA, respectively. It has been demonstrated in an earlier work 30 that by increasing the initial temperature, the material formability was significantly improved without adversing the grain refinement effect, which could be completed in the following steps. In other words, the strategy of performing a first set of passes at relatively high temperature aimed at achieving a first substantial grain-size reduction and then completing the refinement in the required regime at lower temperature by subsequent set of passes revealed to be successful. [fig_ref] Figure 2: Microstructure of the samples after eCAp [/fig_ref] shows the microstructure of ME. It is apparent that the original coarse grained structure was significantly refined (compare with [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref] , twinning was evident in the coarser grains also of the ECAP treated samples. The process was interrupted after 4 passes at 250 °C due to failure of specimens, preventing the achievement of a finer structure. Eventually, an equiaxed structure with grain size of 22 μm was achieved by ECAP in ME samples. In contrast, by a careful selection of ECAP processing temperatures and number of passes, a completely recrystallized structure by ultra-fine grains throughout the billet volume of the AE samples was achieved. The average grain size was of about 0.7 µm, as depicted in [fig_ref] Figure 2: Microstructure of the samples after eCAp [/fig_ref]. Mechanical properties Microhardness [fig_ref] Figure 3: Average hardness of the investigated samples before and after eCAp [/fig_ref] Shows the average Vickers microhardness measured on longitudinal sections of the samples before and after ECAP. It is clearly demonstrated that for pure Mg the hardness values remained almost constant with no significant improvement after ECAP processing. However, it is observed that for the ZK60 alloy investigated, hardness rose through ECAP, resulting in an improvement of about 18% (sample AE). ## Tensile properties The tensile stress vs. strain curves for as-received and ECAP processed samples are depicted in [fig_ref] Figure 4: tensile stress-strain curves measured at room temperature of the as-received and the... [/fig_ref]. Tensile properties including, 0.2% proof stress (YS), ultimate tensile strength (UTS) and fracture elongation values (FE) drawn from these curves are also plotted in [fig_ref] Figure 5: tensile properties of Mg and ZK60 alloy as a function of samples... [/fig_ref]. The data demonstrate that for the pure Mg (MA and ME samples, see Figs. 4A and 5) the yield stress did not change significantly after ECAP, while the UTS improved from 285 MPa up to a value of 326 MPa and a substantial increment of fracture elongation was measured as well, from an initial value of 4.4% to 10.3%. As shown in [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref] , the grain size was considerably reduced by performing ECAP. Nevertheless, it is supposed that such grain size dependency is not the only factor influencing the yield strength and fracture elongation properties in Mg alloys. It is believed that mechanical behavior of the ECAP processed Mg would also be related to crystallographic texture modification in accordance with other literature reports. [bib_ref] Texture development and its effect on mechanical properties of an AZ61 Mg..., Kim [/bib_ref] [bib_ref] The activity of nonbasal slip systems and dynamic recovery at room temperature..., Koike [/bib_ref] In particular, when comparing the data of AA and AE materials it can be observed that the AE samples, despite their much finer grains featured lower YS and UTS than the as received alloy. It is suggested that this can be related to the dominant texture-softening effect over the grain size strengthening induced during ECAP passes at high temperatures. [bib_ref] Enhanced ductility in strongly textured magnesium produced by equal channel angular processing, Agnew [/bib_ref] On this basis, the elongation to failure of AE remarkably increased with an improvement of 100%, whereas the alloy still kept a relatively high tensile strength required for the stent application (Figs. 4B and 5). [bib_ref] Coronary stents: a materials perspective, Mani [/bib_ref] Extrusion The ECAP processed samples, ME and AE, were then subjected to warm extrusion tests aimed at producing mini-tubes required for stent manufacturing. In order to evaluate the effect of grain refinement induced by ECAP on the mechanical properties of the final tubes and to comparatively evaluate the formability of the UFG materials, MA and AA were also processed as references for the extrusion tests. It is verified that the high formability properties highlighted by the low extrusion load and absence of cracks are often inherited in UFG Mg alloys by the contribution of superplastic effects induced by the grain refinement. [bib_ref] Developing superplasticity in a magnesium alloy through a combination of extrusion and..., Matsubara [/bib_ref] [bib_ref] A two-step processing route for achieving a superplastic forming capability in dilute..., Horita [/bib_ref] Accordingly, extrusion for MEE could be performed at 230°C, while MAE, due to its coarser grain structure and lower formability could be safely extruded only at 300 °C. As a result, of the lower processing temperature, as shown in [fig_ref] Figure 6: Representative micrographs of the mini-tube grain structure after extrusion for Mg and... [/fig_ref] , MEE sample shows much finer grain structure than MAE so that the combination of ECAP and low-temperature extrusion brought about a remarkable grain refinement in pure Mg to approximately 11µm. For the ZK60 alloy, as shown in [fig_ref] Figure 4: tensile stress-strain curves measured at room temperature of the as-received and the... [/fig_ref] , both conditions (AA and AE) featured a high plastic formability. Thus, AEE and AAE were successfully produced even at lower temperatures (150°C). [fig_ref] Figure 6: Representative micrographs of the mini-tube grain structure after extrusion for Mg and... [/fig_ref] suggests that in AAE sample a more homogeneous and finer equiaxed grain structure than in sample AA was obtained. It is worthy to note that no obvious grain growth was observed in AEE, certifying that UFG structure induced by ECAP process was maintained almost stable even after the extrusion at 150 °C [fig_ref] Figure 6: Representative micrographs of the mini-tube grain structure after extrusion for Mg and... [/fig_ref]. For small-size tubes the mechanical properties were characterized by micro-hardness due to geometrical limitation of the samples. [fig_ref] Figure 7: Micro-hardness profiles on longitudinal cross sections of samples subjected to interrupted extrusion... [/fig_ref] shows the hardness profile measured along the cross section parallel to the extrusion direction of interrupted extrusion tests for Mg and ZK60 alloy samples. All curves indicate a gradual increase in hardness from billet region to extruded tube region. From [fig_ref] Figure 7: Micro-hardness profiles on longitudinal cross sections of samples subjected to interrupted extrusion... [/fig_ref] and B it can be understood that during the extrusion of AEE and MEE samples, when the billets were kept inside the die at high temperature a drop in hardness occurred when compared with values of ECAP processed billets. However, this gap was totally counterbalanced by the effect of plastic deformation during extrusion. Moreover, AEE and MEE samples always showed higher hardness values in all tested regions than of the corresponding AAE and MAE samples confirming that the improved properties inherited from ECAP are also kept after extrusion. Corrosion Electrochemical studies [fig_ref] Figure 8: the corrosion potential of pure Mg and ZK60 alloy with time in... [/fig_ref] shows the OCP measurement results of AA, AE, MA, and ME samples. As can be seen by the OCP plot, establishment of the steady-state did not happen before 12 h for pure Mg samples, based on which observation, an extra time was allowed (6 h) to ascertain a steady trend. For alloyed samples however, steady manner was established faster, within four hours. To keep the uniformity in all experiments, an 18 h period was allowed for OCP measurement of all samples. Two distinct levels of OCP values can be recognized according to this plot referring to the composition of the metals. More precisely, a steady-state OCP was observed in the range of -1.7 V vs. Ag/AgCl for pure Mg samples and a second level was observed in the range of -1.5 V vs. Ag/AgCl for ZK60 alloy samples. The 200 mV shift toward more positive potentials reveals a nobler behavior of the alloyed samples, that is believed to be caused by the formation of a protective film of hydroxyapatite (HA) and other Mg/Ca phosphates in SBF. [bib_ref] Research on an Mg-Zn alloy as a degradable biomaterial, Zhang [/bib_ref] Apart from the obvious difference between the OCP of alloyed and pure Mg, the as-received samples in both cases showed a slightly nobler behavior when compared with ECAP samples (OCP AA : -1.49V vs. OCP AE : -1.51 V Ag/AgCl and OCP MA : -1.70V vs. OCP ME : -1.725 V Ag/AgCl ). [fig_ref] Figure 9: potentiodynamic polarization curves of pure Mg and ZK60 alloy immersed in pBS... [/fig_ref] shows the the potentiodynamic measurements of all the investigated samples. The same big distinction observed in OCP results between the alloyed and pure Mg is present here in inversion potentials of the two groups as well. Another significant difference between these two groups of materials can be recognized in the presence of a breakdown potential in the anodic parts of MA and ME samples that is lacking in AA and AE samples. This potential (-1.5 V Ag/AgCl for both MA and ME) at which a sudden "jump" in the corrosion current is observed following a passive-like limited current region, is another sign of the formation of a "protective" layer, that is then broken down at this "pitting" potential. [bib_ref] Corrosion behaviour of AZ21, AZ501 and AZ91 in sodium chloride, Song [/bib_ref] [bib_ref] Zainal Abidin NI. Corrosion mechanism applicable to biodegradable magnesium implants, Atrens [/bib_ref] In the case of AA and AE samples however, it is seen that the anodic region already started from this pitting potential and hence, the jump is not observed on the curves. In other words, it is supposed that the inversion potential of AA and AE samples roughly coincides with the pitting potential. It is reported in the literature 45 that one of the effects of Zn alloying element on Mg consists of localized corrosion, in the form of pitting corrosion. This observation is in good agreement with the aforementioned anodic behavior observed in the ZK60 samples. Regarding the corrosion current densities (i corr ) estimated from the Tafel extrapolation method, AA and AE samples were found to show similar i corr values, of 60.4 and 56.2 μA cm -2 , respectively. These data are higher than those of MA and ME samples (both showing an i corr of 44.7 μA cm -2 ) suggesting that, although the protective film is more likely to be formed in the alloyed samples, the breakdown of is this layer is inevitable in chlorine-containing environments. Therefore, not only the permanent protection is not provided, but even a faster localized corrosion would happen upon the onset of pitting. Nevertheless, in this case, the gap between the i corr of alloyed and unalloyed samples is not significant. On the other hand, the effect of ECAP treatment on i corr of each group of the samples seems to be modest. Even when considering the MA and ME samples featuring an inversion potential difference of about 100mV (-1.72 and -1.62 V Ag/AgCl for ME and MA, respectively), i corr remains almost constant at 44.7 μA cm -2 . Corrosion morphology [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref] depict the surface views of the investigated samples after 96 h immersion in PBS solution and following the removal of corrosion products by chromic acid. In both rod and tube samples, a clear difference can be highlighted between the as-received samples of pure Mg and ZK60 alloy (MA vs. AA and MAE vs. AAE). Referring to the discussions given section 3.4.1, increased pitting corrosion is confirmed from surface aspect as well. Moreover, while in case of pure Mg, ECAP treatment does not seem to have a noticeable effect on the corroded microstructure (compare MA to ME in [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref] and MAE to MEE in [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref] , for the ZK60 alloy samples, the ECAP effect on the corrosion regime and microstructure seems remarkable (compare AA to AE in [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref] and AAE to AEE in [fig_ref] Figure 1: Microstructure of starting materials [/fig_ref]. This phenomenon can be elucidated by considering the role of alloying elements and the related formation of the second phases. Due to the larger size of intermetallic particles in AA and AAE samples and lower homogeneity for their dispersion in the matrix compared with the ECAP processed samples, it can be reasonably supposed that the micro-galvanic damage is more active in AA and AAE samples, [bib_ref] Zainal Abidin NI. Corrosion mechanism applicable to biodegradable magnesium implants, Atrens [/bib_ref] causing the formation deeper corrosion pits. [bib_ref] Corrosion Mechanisms of Magnesium Alloys, Song [/bib_ref] [bib_ref] Corrosion of an extruded magnesium alloy ZK60 component-The role of microstructural features, Zeng [/bib_ref] On the contrary when a more homogeneous dispersion of the second phase particles arising from the severe plastic deformation induced by ECAP is promoted, a uniform general corrosion regime can occur preferentially over the localized corrosion mechanism. ## Experimental procedure ## Materials and ecap processing In this investigation, commercial ZK60 (Mg-5.3Zn-0.48Zr, by wt. %) extruded alloy and hot rolled pure magnesium (99.94%) were selected. Cylindrical specimens of 10 mm in diameter with length of 100 mm were machined from the starting materials. The die used for ECAP processing possessed two channels intersecting at an angle of 110° and with an angle of 20° representing the outer arc of curvature. According to well established Iwahashi equation, 48 this geometry generates an equivalent plastic strain of 0.76 per each pass. The whole die was uniformly heated by 4 electrical resistance heaters distributed along the vertical channel and the temperature was monitored at the intersection points of the channels. All multiple pressings were executed by always rotating the sample in the same direction along the longitudinal axis by 90° before each new pass (route Bc according to established designation given in the literature [bib_ref] Equal channel angular extrusion: from macromechanics to structure formation, Segal [/bib_ref]. Samples were sprayed with MoS 2 lubricant and pressed into the ECAP die at a speed of 30 mm/min. For ZK60 alloy, a first set of ECAP passes was performed at 250 °C for up to 4 passes. Selected specimens were additionally subjected to a second and third set of pressings conducted at 200 °C and 150 °C for other additional 4 passes, at each temperature. For pure magnesium, the first set was performed at 275 °C for 2 passes followed by a second set at 250 °C for 4 passes. Further processing at lower temperature led to extensive cracking and it was therefore abandoned. Extrusion Extrusion of ECAP processed materials was performed by using a laboratory device already adopted for coarse grained alloys, described in details in a previous work.Extruded minitubes were produced from the ECAP processed billets at temperatures of 150 °C and 230 °C for ZK60 alloy and pure Mg, respectively. The extruded tubes had an outer diameter of 4 mm and an inner diameter of 2.5 mm, corresponding to a reduction ratio of 9:1 performed at a ram speed of 5 mm/min and 2 mm/ min for ZK60 and pure Mg, respectively. In order to compare the effects of ECAP on properties of the small size tubes, reference samples of the as received ZK60 alloy and pure Mg were directly extruded at 150 °C and 300 °C, respectively. [fig_ref] Table 1: eCAp and extrusion processing conditions of the samples investigated [/fig_ref] summarizes the conditions investigated for both materials at the different ECAP and extrusion temperatures. ## Microstructural characterization The specimens for microstructure observation were longitudinally cut (parallel to the ECAP and extrusion direction) ground and polished according to standard metallographic procedures. The polished specimens were etched in a solution of 6 g picric acid, 5 ml acetic acid, 10 ml water and 100 ml ethanol. The microstructures of the samples were observed by optical microscopy (OM) and field emission gun scanning electron microscopy (FEG-SEM) on longitudinally cross-sectioned samples after ECAP and extrusion. Characterization of the extruded tubes was comparatively evaluated considering samples extracted from the die after interrupted extrusion trials, where the billet region (thermal effect due to holding at high temperature) and the tube region (combined effects of high temperature and plastic deformation) could be analyzed. The average grain size was evaluated following the procedure given in ASTM E112 standard for the linear intercept method. ## Mechanical characterization Mechanical properties after ECAP process were evaluated by tensile and Vickers microhardness tests. Tensile specimens were machined along the ECAP direction with a gage length of 12 mm and diameter of 4 mm. tensile tests were performed at room temperature at a strain rate of 10 -3 s -1 . Microhardness measurements were made on planes parallel to the extrusion and ECAP directions with an indenter load of 1N. Due to size limitations, the mechanical properties of the extruded tubes were characterized only by Vickers microhardness tests. Measurements were performed on the same samples of the interrupted extrusion trials used for microstructural characterization, where both billet region and tube region could be tested. ## Electrochemical measurements The electrochemical corrosion behavior of MA, AA, ME, and AE samples was studied through open circuit potential (OCP) and potentiodynamic polarization tests. A PTFE sample holder with a circular opening of 5.5 mm diameter was used to isolate the body of the sample from the electrolyte and to provide an electrode area of 23.8 mm 2 in contact with the electrolyte. A phosphate buffer solution (PBS) maintained at 37 °C and pH 7.4 was selected as the electrolyte simulating the body fluid. A three electrode set up comprised a platinum coated titanium sheet as counter electrode, an Ag/AgCl, Sat. KCl, reference electrode, and different magnesium samples as working # Conclusion The microstructure, mechanical and corrosion properties of ultra-fine grained ZK60 alloy and of pure Mg were investigated in view of the potential application of these materials for biodegradable devices. The conclusion can be summarized as follows: (1) ECAP process was performed according to a multi-step strategy aimed at achieving a first refining of the structure and then reaching the UFG grain size range by still reducing the processing temperature. A homogeneous equiaxed grain structure with average size in the range of 700 nm was successfully achieved for the ZK60 alloy, while dynamic recrystallization and rapid grain coarsening occurring in commercial pure magnesium prevented the achievement of a grain size smaller than 22 μm. (2) ECAP process had a considerable influence on the grain size as well as on texture of pure Mg and ZK60 alloy. A combination of texture softening and grain refinement in ECAP processed samples led to excellent mechanical properties with about 100 and 135% improvement in fracture elongation for ZK60 and pure Mg, respectively, while keeping relatively high TYS and UTS values. (3) ECAP processed ZK60 and pure Mg billets showed better plastic formability than non-ECAP treated samples and therefore, were successfully extruded at 150 and 230 °C, to produce small-size tubes as stent precursors. The UFG structure of ZK60 alloy was maintained also after extrusion due to the contribution of dynamic recrystallization and low processing temperature. At the beginning of the extrusion process, hardness of pure Mg and of ZK60 alloy dropped by warm temperature holding, but was soon recovered due to the refinement promoted by dynamic recrystallization at low processing temperature, (4) Based on the OCP and potentiodynamic experiments, alloying elements have the substantial effect of modifying the protective surface layer on the free corrosion potential and corrosion current density. This effect provides a sort of nobility action on samples, but second-phase particles formed by the presence of alloying elements also encourage the localized corrosion regime that leads to higher corrosion rates and deep local corrosion damage. ECAP treatment had minor effect on electrochemical measurements of OCP and potentiodynamic tests. However, its main contribution is on the restrictions put on the heterogeneity of microstructure to make a finer and more homogeneous second-phase dispersion and thereby, depressing the localized corrosion pitting. ## Disclosure of potential conflicts of interest No potential conflicts of interest were disclosed. [fig] Figure 1: Microstructure of starting materials: MA (A) and AA (B). [/fig] [fig] Figure 2: Microstructure of the samples after eCAp: Me (A) and Ae (B). [/fig] [fig] Figure 3: Average hardness of the investigated samples before and after eCAp. [/fig] [fig] Figure 4: tensile stress-strain curves measured at room temperature of the as-received and the eCAp processed Mg (A) and ZK60 alloy (B). [/fig] [fig] Figure 5: tensile properties of Mg and ZK60 alloy as a function of samples condition. YS: tensile yield strength; UtS: ultimate tensile strength; Fe: fracture elongation. [/fig] [fig] Figure 7: Micro-hardness profiles on longitudinal cross sections of samples subjected to interrupted extrusion tests: Mg (A) and ZK60 alloy (B). [/fig] [fig] Figure 6: Representative micrographs of the mini-tube grain structure after extrusion for Mg and ZK60alloy at different temperatures. (A) MAe at 300 °C, (B) Mee at 230 °C, (C) AAe at 150 °C, (D) Aee at 150 °C. [/fig] [fig] Figure 8: the corrosion potential of pure Mg and ZK60 alloy with time in pBS at 37 °C. [/fig] [fig] Figure 9: potentiodynamic polarization curves of pure Mg and ZK60 alloy immersed in pBS before and after eCAp. [/fig] [table] Table 1: eCAp and extrusion processing conditions of the samples investigated. MA As-received pure Mg Me MA eCAp treated for 2 passes at 275 ∘C+ 4 passes at 250 ∘C Ae AA eCAp treated for 3 steps of 4 passes at namely, 250 ∘C + 200 ∘C + 150 ∘C Aee Ae + extrusion at 150 ∘C AAe AA + extrusion at 150 ∘C [/table]

Extra-Gastrointestinal Stromal Tumor (EGIST) in the Pelvis Mimicking Retroperitoneal Sarcoma Gastrointestinal stromal tumors (GISTs) are among the most common mesenchymal tumors 1 of the gastrointestinal (GI) tract; they account for approximately 3% of all GI tumors, with up to 70% found in the stomach, 25% in the small intestine, 5% in the colon and rectum, and 5% in the esophagus. 2 The cell that gives rise to these tumors is a pluripotent mesenchymal stem cell that has been automated to differentiate into interstitial Cajal cells, 2 which are found in intestinal wall, 3 and regulate its peristalsis. 2 Extra-gastrointestinal stromal tumors (EGISTs), as the term implies, are GISTs found outside the GI tract, 2,3 representing for less than 5% of total GISTs, 3 and less than 1% of total GI tumors. 2 EGISTs are most commonly found in the omentum, mesentery, retroperitoneum, pelvic region, abdominal wall, liver, gallbladder, pancreas, bladder, seminal vesicle, vagina prostate, 3 and scrotum. 2 The majority of cases involve elderly patients, ranging from 45.8 to 59 years as mean age and are larger than GISTs at the time of diagnosis. 1 EGISTs share histopathological and molecular characteristics with GISTs, 2 but unlike GISTs, some research has shown that EGISTs are derived from Cajal cells or pluripotent stem cells found outside the GI tract. 3 Histopathologically, they are divided into spindle, epithelioid, and mixed types. 1 Abstract Extra-gastrointestinal stromal tumors (EGISTs) are rare mesenchymal tumors accounting for less than 1% of total gastrointestinal tumors. They tend to be aggressive and have a poor prognosis. Unfortunately, there is a lack of data or controversial data due to its scarcity. Therefore, we report a case of pelvic EGIST misdiagnosed as retroperitoneal sarcoma. We opted for surgical management followed by adjuvant oral chemotherapy with imatinib. Ã These authors contributed equally to this work. Gastrointestinal stromal tumors (GISTs) are among the most common mesenchymal tumors 1 of the gastrointestinal (GI) tract; they account for approximately 3% of all GI tumors, with up to 70% found in the stomach, 25% in the small intestine, 5% in the colon and rectum, and 5% in the esophagus. [bib_ref] Perirectal extragastrointestinal stromal tumor: an unusual presentation, Elagami [/bib_ref] The cell that gives rise to these tumors is a pluripotent mesenchymal stem cell that has been automated to differentiate into interstitial Cajal cells, 2 which are found in intestinal wall, [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] and regulate its peristalsis. [bib_ref] Perirectal extragastrointestinal stromal tumor: an unusual presentation, Elagami [/bib_ref] Extra-gastrointestinal stromal tumors (EGISTs), as the term implies, are GISTs found outside the GI tract, [bib_ref] Perirectal extragastrointestinal stromal tumor: an unusual presentation, Elagami [/bib_ref] [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] representing for less than 5% of total GISTs, [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] and less than 1% of total GI tumors. [bib_ref] Perirectal extragastrointestinal stromal tumor: an unusual presentation, Elagami [/bib_ref] EGISTs are most commonly found in the omentum, mesentery, retroperitoneum, pelvic region, abdominal wall, liver, gallbladder, pancreas, bladder, seminal vesicle, vagina prostate, 3 and scrotum. [bib_ref] Perirectal extragastrointestinal stromal tumor: an unusual presentation, Elagami [/bib_ref] The majority of cases involve elderly patients, ranging from 45.8 to 59 years as mean age and are larger than GISTs at the time of diagnosis. [bib_ref] Primary extra gastrointestinal stromal tumors of the abdomen, Uzunoglu [/bib_ref] EGISTs share histopathological and molecular characteristics with GISTs, 2 but unlike GISTs, some research has shown that EGISTs are derived from Cajal cells or pluripotent stem cells found outside the GI tract. [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] Histopathologically, they are divided into spindle, epithelioid, and mixed types. 1 ## Keywords [formula] ► extra-gastrointestinal ► stromal tumors ► GIST ► retroperitoneal sarcoma ► surgery ► case report [/formula] # Abstract Extra-gastrointestinal stromal tumors (EGISTs) are rare mesenchymal tumors accounting for less than 1% of total gastrointestinal tumors. They tend to be aggressive and have a poor prognosis. Unfortunately, there is a lack of data or controversial data due to its scarcity. Therefore, we report a case of pelvic EGIST misdiagnosed as retroperitoneal sarcoma. We opted for surgical management followed by adjuvant oral chemotherapy with imatinib. There is a lack of data or controversial data because it is a rare pathology and little studied as an independent entity. [bib_ref] Primary extra gastrointestinal stromal tumors of the abdomen, Uzunoglu [/bib_ref] The exact incidence of EGISTs and their tumor behavior is unknown. [bib_ref] Perirectal extragastrointestinal stromal tumor: an unusual presentation, Elagami [/bib_ref] Here, we present a case of EGIST found incidentally in an elderly patient, who was surgically treated and followed by adjuvant oral chemotherapy. ## Case description We present a case of 75-year-old male patient, with an unremarkable medical history, presented to outpatients clinics in another institution for 2 months history of urinary tract symptoms including dysuria, incontinence, and small volume of urine when he voids. A urinary tract infection was suspected and the patient was, therefore, treated with a course of antibiotics. Unfortunately, the antibiotic therapy did not alleviate the patient's symptoms, so he presented to our clinics. A palpable soft mass at the lateral wall of the rectum was found on physical examination. A colonoscopy was performed, and no abnormal findings were found. Subsequently, the pelvic magnetic resonance imaging revealed a 14.5 Â 8.6 Â 8.1cm pelvic heterogeneous exophytic mass that shows heterogeneous T2, mixed hyperand hypo-T1 signal intensities (denoting hemorrhagic components), heterogeneously enhanced and demonstrating areas of restricted diffusion on diffusion-weighted imaging (►Figs. 1-3). It shows mass effect on the rectum that is compressed to the left with invasion of the mesorectal fascia and the pelvic side walls. It also indents the posterior border of the prostate (►Fig. 4). Subcentimetric pelvic retroperitoneal lymph nodes are seen, the largest seen in the right internal iliac chain measuring 6 mm. The mass biopsy was inconclusive with the suspicion of having a retroperitoneal sarcoma as one of the differential diagnoses; hence, we opted for surgical resection. On the day of the surgery, a bilateral double J stent was put in place. Laparoscopically, exploration and dissection through the pelvic cavity revealed the presence of the mass that was in firm contact with the posterior part of the bladder, the right ureter, and the mesorectum. Invasion of these structures could not be ruled out by laparoscopy; therefore, conversion to open surgery was performed, where total tumor excision was performed along with partial cystectomy and partial excision of the mesorectum. A protective loop ileostomy was performed for fear of having iatrogenic or ischemic rectal lesions after the operation. The patient tolerated the surgery well and was subsequently discharged home without major complications. The pathology report and the immunochemistry study confirmed the diagnosis of GIST, by the diffuse expression of CD117 as well as discovered on GIST-1 (DOG1; ►Fig. 5) and h-Caldesmon. In addition, since both spindle and epithelioid cells were distinguished, this tumor was classified as a mixed cell type (►Fig [fig_ref] Figure 6: A [/fig_ref]. Grossly, areas of necrosis and hemorrhages among the epithelioid cells were described (►Fig. 7). The mitotic rate was 3. By combining histology and morphology, this tumor as described corresponded to the criterion of malignant EGIST. The patient started adjuvant chemotherapy with imatinib. In follow-up, he showed a good response, no reported side effects, and no signs of recurrence. # Discussion EGISTs are rare tumors found in the nondigestive tract, 4 which affect more commonly women and the elderly patients. [bib_ref] Primary extra gastrointestinal stromal tumors of the abdomen, Uzunoglu [/bib_ref] They are more commonly seen in larger sizes 1 and advanced stage due to the delay in the diagnosis. [bib_ref] Clinical features of extragastrointestinal stromal tumor compared with gastrointestinal stromal tumor: a..., Feng [/bib_ref] They are identified according to the diagnostic characteristics of GIST (Anapath: abbreviation of Anatomopathological examination, immunohistochemistry, and molecular genetic analysis). There are studies that have suggested that EGIST is a secondary lesion or metastatic deposit of GIST, but others have shown that it is a special entity with its own characteristics. [bib_ref] Clinical features of extragastrointestinal stromal tumor compared with gastrointestinal stromal tumor: a..., Feng [/bib_ref] EGISTs have no specific symptoms or radiological features; the most common presentation is abdominal discomfort or pain, 2 and these vary depending on the tumor size and location. 1 They are discovered incidentally during investigations for abdominal pain, abdominal mass, or during a laparotomy. [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] Patient history, physical examination, appropriate blood work, and imaging studies, as well as surgical evaluation, may be included in the EGIST workup. Endoscopy and endoscopic ultrasound can be helpful in some cases. [bib_ref] Perirectal extragastrointestinal stromal tumor: an unusual presentation, Elagami [/bib_ref] An enhanced computed tomography scan usually reveals some distinguishing features, such as a large mass with peripheral enhancement of unknown origin shifting adjacent structures and possible abdominal lymphadenopathy nodes. [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] Imaging alone does not distinguish EGIST from other abdominal pathologies 3 ; to confirm the diagnosis, a percutaneous transperitoneal biopsy or an endoscopic transmural biopsy is required, 2 and immunohistochemistry studies reveal that the cells are positive for immunological markers such as CD117, DOG1, CD34, B-cell lymphoma 2, desmin, smooth muscle actin, [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] and S100. [bib_ref] Primary extra gastrointestinal stromal tumors of the abdomen, Uzunoglu [/bib_ref] The combination of CD117 and DOG1 improves significantly diagnosis accuracy. [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] The differential diagnosis tends to be broad and includes leiomyosarcoma, liposarcoma, fibrosarcoma, 2,3 solitary fibrous tumor, 2 or lymphoma. [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] The treatment of EGIST is similar to that of GIST, which consists of radical surgical en bloc resection with negative margins. [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] [bib_ref] Clinical features of extragastrointestinal stromal tumor compared with gastrointestinal stromal tumor: a..., Feng [/bib_ref] The efficacy of combination with imatinib as neoadjuvant or adjuvant treatment remains controversial; however, it is recommended for the treatment of unresectable tumors, with dose modification according to the disease progression. [bib_ref] Curative intent treatment of late presented extragastrointestinal stromal tumor: two identical case..., Kadel [/bib_ref] EGISTs are more aggressive than GISTs, but have a poor prognosis, 1 with 5-year survival rate reaching 48.9%. 2 They have a low mitotic index of 5/50 high-power fields and less, and tend to metastasize in up to 30% of cases. 1 Mitosis, necrosis, and large tumor size are considered high-risk factors for a poorer prognosis. [bib_ref] Clinical features of extragastrointestinal stromal tumor compared with gastrointestinal stromal tumor: a..., Feng [/bib_ref] The recurrence rate has been reported up to 23%. [bib_ref] Primary extra gastrointestinal stromal tumors of the abdomen, Uzunoglu [/bib_ref] Failure to explore clinicopathological differences between EGIST and GIST may have resulted in inappropriate treatment approaches, or it may have contributed to EGIST's poor prognosis. [bib_ref] Clinical features of extragastrointestinal stromal tumor compared with gastrointestinal stromal tumor: a..., Feng [/bib_ref] Long-term follow-up studies are needed to recognize the behavior of this rare neoplastic disease. 2 # Conclusion EGISTs are rare mesenchymal tumors with a poor prognosis. They are discovered incidentally and confirmed by the combination of immunohistochemistry CD117 and DOG1 detection. The clinical presentation can be tricky, so we need to be rigorous and keep this differential diagnosis in mind. We encourage researchers to further explore this pathology to obtain clear guidelines on treatment and outcomes. # Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-forprofit sectors. ## Conflict of interest None declared. ## Informed consent Written informed consent was obtained from the patient for publication of this case report and accompanying images. A copy of the written consent is available for review by the editor-in-chief of this journal on request. [fig] Figure 4: The lesion shows mass effect on the rectum that is compressed to the left with invasion of the mesorectal fascia. It also indents the posterior border of the prostate. [/fig] [fig] Figure 1: The pelvic lesion shows heterogeneous signal on T2 (A) and short tau inversion recovery (B) weighted images (all arrows point to tumor). [/fig] [fig] Figure 2: The pelvic lesion shows hyposignal T1 (A) and enhancement after injection of gadolinium (B) (all arrows point to tumor). [/fig] [fig] Figure 3: The pelvic lesion shows restriction on diffusion and apparent diffusion coefficient sequences. The Surgery Journal Vol. 8 No. 3/2022 © 2022. The Author(s). [/fig] [fig] Figure 6: A) Hematoxylin and eosin stain (HE) showing spindle cells. (B) HE showing epithelioid cells. Original magnification x200. [/fig] [fig] Figure 7: Hematoxylin and eosinx200.2 showing areas of necrosis with epithelioid cells. [/fig] [fig] Figure 5: Immunohistochemistry showing staining for discovered on gastrointestinal stromal tumor 1 protein. Original magnification x200. The Surgery Journal Vol. 8 No. 3/2022 © 2022. The Author(s). [/fig]

Risk factors for male breast cancer. Smm.uaryRisk factors for male breast cancer were investigated in a case-control study of 21 cases and 82 controls admitted to hospital for acute, non-neoplastic, non-hormone-related diseases in the Greater Milan area between 1988 and 1994. More educated men tended to be at higher nrsk of breast cancer, with a multivariate odds ratio (OR) of 2.6 [95% confidence interval (CI) 0.7 -9.4]. The OR was 3.2 (95% CI 1.1 -9.6) for those in the higher social class. Men with no offspring were at higher risk than fathers, with an OR of 5.5 (95% Cl 1.8-16.7). A history of breast cancer in female relatives was reported by two cases and one control, giving an OR of 8.5 (95% CI 1.1-69.0). Cases were somewhat heavier than controls, and significantly taller, with an OR of 5.7 (95% CI 1.6-19.9) for subjects taller than 170cm vs shorter ones. The association with weight, however, decreased after allowance for height, and no difference was observed for body mass index. Socioeconomic correlates and family history are similar to well-assessed risk factors for female breast cancer. The associations with anthropometric measures and childkssness may find an explanation in chromosomal abnormalities, such as Klinefelter's syndrome, or other hormone-related disorders. Male breast cancer is extremely rare, representing less than 1% of all breast cancer and less than 0.1% of all cancer deaths in men [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref]. Consequently, epidemiological data are scant. Some associations have been suggested with sociodemographic characteristics, including a direct gradient with social class (i.e. an increased risk in higher social classes) [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref] , although this remains controversial [bib_ref] Risk factors for male breast cancer: a Franco-Swiss case-control study, Lenfant-Pejovic M-H [/bib_ref] [bib_ref] Breast cancer in men: risk factors with hormonal implications, Db [/bib_ref] ; with marital status, never married men being more frequently affected; and with religion, Jewish men being at highest risk [bib_ref] Risk factors for male breast cancer, Mabuchi K [/bib_ref] [bib_ref] Breast cancer in men: risk factors with hormonal implications, Db [/bib_ref] [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref]. Anthropometric characteristics have been investigated, and body mass index was associated with male breast cancer risk in a case-control study from Los Angeles County [bib_ref] A case-control study of male breast cancer, It [/bib_ref]. Previous breast or testicular disease and gynaecomastia have been related to male breast cancer, and associations are reported with orchiectomy, orchitis, testicular injury, late puberty and infertility [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref]. Klinefelter's syndrome is substantially more common among male breast cancer patients [bib_ref] A case-control study of male breast cancer, It [/bib_ref] [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref]. Possibly as a consequence of Klinefelter's syndrome or hormonal abnormalities, infertility and low fertility have also been associated with male breast cancer [bib_ref] Breast cancer in men: risk factors with hormonal implications, Db [/bib_ref] [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref]. Family history of breast cancer has repeatedly been associated with breast cancer risk in female and in male first-degree relatives [bib_ref] A case-control study of male breast cancer, It [/bib_ref] [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref]. In terms of aetiological mechanisms, high oestrogen levels have been reported as a risk factor for male breast cancer [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref] , and various studies have found higher serum or urinary oestrogen levels in cases than in controls, but not all results were consistent [bib_ref] Plasma estrogens and androgens in male breast cancer, De Giuli G [/bib_ref] [bib_ref] Serum oestradiol-17p, testosterone, luteinizing hormone and follicle-stimulating hormone in males with breast..., Ribeiro Gg [/bib_ref] [bib_ref] The sex-hormone profile of male patients with breast cancer, Nirmul D [/bib_ref] [bib_ref] A case-control study of male breast cancer, It [/bib_ref] [bib_ref] Hormones in male breast cancer, Ballerini P [/bib_ref] [bib_ref] Increased plasma prolactin levels in a group of men with breast cancer...., Olsson H [/bib_ref]. To provide further information on this issue, we report here data from a case-control study on male breast cancer conducted in northern Italy. # Material and mehods Data were derived from a case-control study conducted since 1988, based on a network including the major teaching and general hospitals of the Greater Milan area. Trained interviewers identified and questioned male patients with breast cancer and controls among patients admitted to hospital with similar catchment area for acute, non-neoplastic, non-hormone-related diseases. The present analysis was based on data collected until February 1994. The structured questionnaire included information about sociodemographic and anthropometric characteristics, general lifestyle habits, such as smoking, alcohol and coffee consumption, a problem-oriented medical history, family history of breast and other hormone-related cancers in firstdegree female relatives and frequency of consumption of selected indicator foods. Cases were 21 patients with histologically confirmed incident breast cancer and were aged 34-74 years (median age 60). They had been diagnosed during the year before interview, and were admitted to the National Cancer Institute and the Ospedale Maggiore of Milan, including the four largest teaching and general hospitals in the Greater Milan area. Controls were 82 subjects aged 31-74 years (median age 59), in hospital for acute, non-neoplastic, non-hormonerelated diseases. Of these, 43% were admitted for traumatic diseases, 18% for non-traumatic orthopaedic diseases, 8% for acute surgical conditions and 31% for various other disorders, such as skin, eye, ear, nose and throat conditions. The distribution of cases and controls according to age is shown in. Over 80% of cases and 90% of controls resided in the same region, Lombardy. Less than 5% of subjects approached for interview (cases and controls) refused to participate. Social class was defined using occupation codes, and socioeconomic groups comparable to those of the British Registrar General (Leete and Fox, 1977) were defined. To obtain categones with meaningful numbers, we grouped the six original social classes into two broad categories: the upper one included classes 1, 2, and 3 non-manual, corresponding to professional, managerial and intermediate occupations; the lower one included classes from 3 manual to 5, corresponding to manual skilled, low skilled and unskilled occupations. # Data analysis Odds ratios (ORs), with the corresponding 95% confidence intervals (CIs) (Breslow and Day, 1980) of breast cancer were computed by means of multiple logistic regression equations, including terms for quinquennia of age plus area of residence, number of offspring, family history of breast cancer, social class, and, whenever indicated, height. For multiple levels of exposure, the significance of the linear trend in risk was assessed by comparing the difference between the deviances of the models without and with the term of interest to the chi-square distribution with one degree of freedom [bib_ref] Statistical Mfethods in Cancer Research, Ne [/bib_ref]. # Results Socioeconomic charactenrstics, number of offspring and family history of breast cancer are considered inMore educated men tended to be at higher risk of breast cancer. Subjects reporting 12 or more years of schooling had an OR of 2.6 (95% CI 0.7-9.4) compared with less educated ones. Cases also differed from controls with reference to social class, subjects of higher social classes having an OR of 3.2 (95% CI 1.1-9.6). Men with no children were at higher nsk than fathers, with a more than 5-fold increased nsk (OR 5.5, 95% CI 1.8-16.7). This was not accounted for by differences in marital status between cases and controls, since only 10% of cases and 16% of controls were never married. Family history of breast cancer was reported by two cases and one control, giving an OR of 8.5 (95% CI 1.1-69.0). Anthropometric characteristics are presented in. Cases were somewhat heavier than controls. Compared with subjects weighing less than 70 kg, the OR for men weighing between 70 and 79 kg was 2.1, and for the heaviest group was 3.4. The trend in risk with weight, however, was not significant. Cases also tended to be taller than controls, and only one case (5%) compared with 23 (28%) controls was shorter than 165 cm: 19% of cases and 40% of controls were between 165 and 170 cm. and 76% of cases but only 32% of controls were taller than 170 cm. The OR for the highest group compared with the two lowest ones was 5.7 (95% CI 1.6-19.9). When the two higher groups were separately compared with the first one, the ORs were 3.2 for the second and 12.9 for the third. and the chi-squared for trend was significant. When the risk estimates for height and weight were mutually adjusted. the OR for height was not materially modified (OR = 5.2. 95% CI 1.3-20.4). Conversely, the OR for weight materially decreased after allowance for height. and it was 1.6 (95% CI 0.3-9.5) for individuals weighing 70-79 kg and 1.5 (95% CI 0.3-8.2) for those weighing more than 79 kg. Consequently, body mass index (BMI. Quetelet index, kg m -) was unrelated to the risk of breast cancer. ## Discs_ss This study confirms that male breast cancer has several risk factors similar to those well defined for female breast cancer [bib_ref] Epidemiology of breast cancer. Bailliere's, Boyle P [/bib_ref]. In particular. high social class indicators. no offspring and family history of breast cancer were related to male breast cancer. From a methodological viewpoint. it is unlikely that major information bias influenced the present results. Information and recall bias about family history of breast cancer should be hmited. because information was related only to firstdegree relatives. Possible problems of information bias can be related to anthropometric measures. since self-reported height tends to be systematically overestimated, and weight underestimated [bib_ref] Underestimation of relative weight by use of self-reported height and weight, Stewart Aw [/bib_ref]. It is unlikely, however, that height and weight were systematically and differently reported by cases and controls. and hence that any such potential bias accounts for the strong associations observed. Other variables investigated, including education. occupation and number of offspring. should not be appreciably affected by systematic bias. Selection bias is also unlikely to have noticeably affected the results, since cases and controls were chosen from similar catchment areas and participation was almost complete. The association of socioeconomic indicators with breast cancer was not accounted for by other identified risk factors. An American study observed an excess of more educated individuals among cases [bib_ref] Risk factors for male breast cancer, Mabuchi K [/bib_ref]. but this was not reported in a French-Swiss study [bib_ref] Risk factors for male breast cancer: a Franco-Swiss case-control study, Lenfant-Pejovic M-H [/bib_ref]. whereas in another American study including 227 cases the only indicator of higher social class significantly related to male breast cancer risk was Jewish religion [bib_ref] Breast cancer in men: risk factors with hormonal implications, Db [/bib_ref]. With reference to females. an Italian study. conducted on females in the same area and with the same methodology of the present study. found a significantly increased risk of about 50% for more educated and higher social class women [bib_ref] General epidemiology of breast cancer in Northern Italy, Vecchia [/bib_ref]. In relation to anthropometric measures. the association with height was stronger than that with weight. and the latter was largely accounted for by height. This is consistent with the lack of association with BMI. which is a measure of weight uncorrelated to height [bib_ref] Some mathematical properties of weight-for-height indices used as measures of adiposity, Benn Rt [/bib_ref]. This result suggests that some chromosomal disorder. such as Klinefelter's syndrome, may be at the base of the association. Subjects with Klinefelter's syndrome tend in fact to be taller [bib_ref] Disorders of sexual differentiation, Wilson [/bib_ref]. Alternatively. high stature may be an indicator of a more affluent diet in infancv and childhood. in agreement Awith results regarding women [bib_ref] Breast cancer risk assessed by anthropometry in the NHANES I epidemiological follow-up..., Swanson Ca [/bib_ref]. This issue of anthropometric factors and male breast cancer is. however. still debated. In a large population-based American study. no trend of increasing risk with height was found [bib_ref] Breast cancer in men: risk factors with hormonal implications, Db [/bib_ref]. and in another American study [bib_ref] A case-control study of male breast cancer, It [/bib_ref] body weight was found to be associated with occurrence of male breast cancer. In that study. anywav. cases were only moderately taller than controls. and no reciprocal allowance was made of weight and height. Being childless was associated with an increased risk. Although most cases and controls were married. controls had significantly more children than cases. suggesting an underlying problem of infertility among cases. In other studies. fatherhood was protective against male breast cancer. and the risk decreased with numbers of children fathered [bib_ref] Breast cancer in men: risk factors with hormonal implications, Db [/bib_ref] [bib_ref] Review article: Epidemiology of male breast cancer. A meta-analysis of published case-control..., Sasco J [/bib_ref] Other. more accurate indicators of Klinefelter's syndrome were not available, preventing a thorough assessment of this issue. Other studies [bib_ref] Carcinoma of the breast and Kinefelter's syndrome, Harnden Dg [/bib_ref] [bib_ref] A case-control study of male breast cancer, It [/bib_ref] investigated sex chromatin positivity and the proportion of positive cases was around 3%. Thus, also in this population only a fraction of cases of male breast cancer should be related to Klinefelter's syndrome. which, in turn, is unlikely to account totally for the associations with childlessness and height that emerged. Plasma oestradiol is elevated in subjects with Klinefelter's syndrome. and testosterone is low [bib_ref] Disorders of sexual differentiation, Wilson [/bib_ref]. It has also been suggested that higher oestrogen and lower androgen levels may decrease fertility in the general population [bib_ref] Are oestrogens involved in falling sperm counts and disorders of the reproductive..., Sharpe [/bib_ref]. This offers a plausible pathological link for the relationship of male breast cancer also for some of the other risk factors. including anthropometric measures and number of children. Body weight is thought to be associated with increased oestrogen levels. which in men are mainly derived from aromatisation of testosterone in the adipose tissue. and some studies found elevated oestrogen levels in males with breast cancer [bib_ref] Plasma estrogens and androgens in male breast cancer, De Giuli G [/bib_ref] [bib_ref] Serum oestradiol-17p, testosterone, luteinizing hormone and follicle-stimulating hormone in males with breast..., Ribeiro Gg [/bib_ref] [bib_ref] The sex-hormone profile of male patients with breast cancer, Nirmul D [/bib_ref] [bib_ref] Increased plasma prolactin levels in a group of men with breast cancer...., Olsson H [/bib_ref]. although the issue is still unsettled [bib_ref] A case-control study of male breast cancer, It [/bib_ref]. Although the limited number of cases and the uncertainties in the interpretations preclude definite conclusions. this studv provides some support to the indications that male and female breast cancer share at least some aetiological factors. This is also reflected in the biological characteristics of male breast cancer. whose prognosis and survival appear to be similar to its much more common female counterpart [bib_ref] The survival pattern of male breast cancer. An analysis of 1429 patients..., Ho [/bib_ref]. [table] Table n: Distribution of 21 cases of male breast cancer and 82 controls according to sociodemographic factors and family history, and corresponding odds ratios (with aEstimates from multiple logistic regression equations including terms for age, height and the above variables. bReference category. 'Including classes from 3 manual to 5 of the British Registrar's General Classification. [/table] [table] Table m: Distribution of 21 cases of male breast cancer and 82 controls according to [/table]

Chronic capsiate supplementation increases fat-free mass and upper body strength but not the inflammatory response to resistance exercise in young untrained men: a randomized, placebo-controlled and double-blind study Background: Acute capsaicinoid and capsinoid supplementation has endurance and resistance exercise benefits; however, if these short-term performance benefits translate into chronic benefits when combined with resistance training is currently unknown. This study investigated changes of chronic Capsiate supplementation on muscular adaptations, inflammatory response and performance in untrained men. Methods: Twenty untrained men were randomized to ingest 12 mg Capsiate (CAP) or placebo in a parallel, double-blind design. Body composition and performance were measured at pre-training and after 6 weeks of resistance training. An acute resistance exercise session test was performed pre and post-intervention. Blood samples were collected at rest and post-resistance exercise to analyze Tumor necrosis factor-(TNF-), Soluble TNFreceptor (sTNF-r), Interleukin-6 (IL-6) and Interleukin-10 (IL-10). Results: Exercise and CAP supplementation increased fat-free mass in comparison to baseline by 1.5 kg (P < 0.001), however, the majority of the increase (1.0 kg) resulted from an increase in total body water. The CAP change scores for fat-free mass were significantly greater in comparison to the placebo (CAP Δ%= 2.1 ± 1.8 %, PLA Δ%= 0.7 ± 1.3 %, P = 0.043) and there was a significant difference between groups in the bench press exercise (P = 0.034) with greater upper body strength change score for CAP (Δ%= 13.4 ± 9.1 %) compared to placebo (Δ%= 5.8 ± 5.2 %), P = 0.041. CAP had no effect on lower body strength and no supplementation interactions were observed for all cytokines in response to acute resistance exercise (P > 0.05). Conclusion: Chronic Capsiate supplementation combined with resistance training during short period (6 weeks) increased fat-free mass and upper body strength but not inflammatory response and performance in young untrained men. Keywords: Pre-workout, Hypertrophy, Performance, Strength training Background Capsaicin is a bioactive phytochemical compound found primarily in chili peppers and other spicy foods, and has been purported to increase metabolism, muscular performance, relieve pain, and improve the immune system function. Capsiate (CAP) is chemical analogs of capsaicin with an ester linkage replacing the amide bond between the vanillyl moiety and the fatty acid chain. In humans, acute CAP supplementation increased the total volume of exercise during an acute resistance exercise session performed to muscular failure and decreased the rate of perceived exertion in a trained population. Similar findings of increased muscle performance were also observed in mice, where chronic capsaicin supplementation increased forelimb grip strength in a dose dependent manner. Although the mechanisms responsible for increased muscle performance are not fully elucidated in skeletal muscle, capsaicin has been shown to activate the transient receptor potential vanilloid-1 (TRPV1), thus increasing the release of calcium (Ca + ) by the sarcoplasmic reticulum. Given the crucial role that that Ca + ions exert on muscle contraction, such increases may also positively influence force generation. Exercise training has been shown to induce profound changes in muscle physiology. For example, during muscle contractions, IL-6 gene expression is activated by Ca + dependent pathways, resulting in a transient, but potent increased IL-6 secretion. In this regard, Obi et al.demonstrated that capsaicin was able to activate the TRPV1 in myoblasts leading to an increase in interleukin-6 (IL-6) mRNA expression. The importance of increased systemic IL-6 for the immune system relies in its potential anti-inflammatory response. Increased IL-6 secretion by contracting skeletal muscles has been demonstrated to inhibit tumor necrosis factor alpha (TNF-a) and increases the release of IL-10 by immune cells. In fact, in a previous study conducted by , resistance training was capable to increase the IL-10/TNF-a ratio in muscle samples from rat muscle. However, since muscle samples also contain other cells types, the exact source responsible for such changes were not evaluated. Since capsaicin/CAP increases muscle IL-6 mRNA gene expression, and since exercise training is a potent stimulus for IL-6 secretion, it is reasonable to hypothesize that both strategies may present synergistic anti-inflammatory effects. Moreover, since acute capsaicin/CAP increases muscle performanceand energy expenditure, these effects may summate when supplemented during chronic exercise training, thus promoting additional adaptations. Hypothetically, such benefits will manifest not only in the immune system, but also in the muscle performance and perhaps, in body composition. Therefore, the main objective of the present study was to test if CAP supplemented before resistance training sessions would be capable to summate and produce a chronic synergistic effect. Specifically, reduced inflammatory response, improved anti-inflammatory response, increased muscle performance and enhanced body composition are hypothesized to occur in a CAP plus resistance training group compared with a placebo plus resistance training mainly in untrained subjects, since they could exhibit greater inflammation than trained subjects using the same load and maybe higher response with intervention. If confirmed, CAP could be utilized in combination with resistance training to potentiate the already known benefits of resistance training, in the supra-cited variables. # Methods ## Experimental design Firstly, the participants completed two weeks of familiarization with the exercise program routine. On the first visit to the laboratory, the participants were assessed for anthropometrics and body composition. On the second visit, 24 h later, the one repetition maximum test (1RM) was performed. Next, participants were pairmatched based on initial fat-free mass, fat mass and strength levels, and then randomly allocated to one of two treatment groups (CAP or Placebo). Food records were distributed to all participants pre and postintervention to record food intake for three nonconsecutive days (one weekend and two weekdays), where one weekday corresponded to the day before the acute resistance exercise session. The third visit was separated by 72 h and subjects performed the acute resistance exercise session test. Blood samples were collected 90 min post-prandial (rest) and immediately following the acute resistance exercise session test. After 48 h, both groups participated in a 6-week progressive resistance training program, as shown in Supplementary Table 1, combined with placebo or CAP supplementation, and then returned to laboratory at post-training to repeat all assessments (4th to 6th visits). Supplementation continued through the post-training evaluation phase. The experimental design is illustrated in. ## Participants This randomized, placebo-controlled and double-blind study was carried out according to the 2013 Revision of the Declaration of Helsinki and it was approved by the Institutional Review Board from Federal University of Piaui in February, 2019 (Protocol number: 3.169.545) according the ethical standards. Participants were invited through media and newspaper advertising at campus to participate in the study. The participants contacted the researchers by phone and an appointment was made in order to carry out a more detailed interview. The inclusion criteria for the present study were: (1) males between 18 and 30 years of age; (2) had not participated in regimented resistance training in the previous 6 months; (3) had not used any ergogenic supplement for at least two years prior to the study; (4) did not smoke or drink alcohol within of testing visits (1st to 3rd visit at baseline and 4th to 6th visit at post-training); (5) no contraindications involving the cardiovascular system, muscles, joints, or bones of the lower and upper limbs that could limit exercise. All participants signed a consent form and were informed about the purpose of the study and the possible risks. During the study, all participants were instructed not to use any other supplement or ergogenic substance and to maintain their current diets. Additional exclusions criteria included missing more than 3 workouts during the intervention. Out of a total of 58 men who participated in the first screening, 28 met all the inclusion criteria and 22 agreed to participate in the study protocol. This study used a randomized, double-blind design. Participants were pairmatched based on initial fat-free mass, fat mass and strength levels and then randomly allocated to one of two treatment groups: CAP (n = 11) and placebo (n = 11). During the intervention, one participant from placebo group dropped out of the study due to unspecified reasons and there was an outlier in the same group who was excluded from the final analysis due higher fat mass in relation to sample. It was calculated as the ratio Z as the difference between the outlier and the mean divided by the SD (via (https://www.graphpad.com/quickcalcs/ grubbs1/) and the outlier comes from a different population if Z is greater than 1.96. Thus, the final sample analyzed was 11 subjects in the CAP group and 9 in the placebo group. ## Procedures Dietary intake assessment and supplementation procedure Food records were distributed to all participants pre and post-intervention to record food intake for three nonconsecutive days (one weekend and two weekdays), where one weekday corresponded to the day before the acute resistance exercise session. A breakfast (total energy 603 ± 65 kcal, consisting of 25 % protein, 50 % carbohydrates and 25 % fat) was provided to participants before the acute resistance exercise (1 h 30 min). The software Dietbox (version 3.3.0) utilized the database of Brazilian food composition table (TACO) to calculate dietary intake. The volunteers were instructed not to consume chili peppers or other spicy foods and any kind of supplementation during the study, as well as coffee, tea, alcohol and/or stimulant drinks for a period of 12 h prior to assessments. Each participant randomly consumed 2 capsules of placebo (starch) or CAP with 6 mg Capsiate per capsule (12 mg Capsiate in total), which were identical in appearance to ensure a double-blind design, 45 min before each experimental test and before lunch on non-training days. This time was selected because capsaicin reaches maximum concentrations within 45 min after ingestion. CAP was standardized to contain 50 % Capsiate (Capsicum annuum L.) (Purifarma-Gemini Pharmaceutical Industry Ltda, Anapolis, GO, Brazil). Study products were delivered to each individual subject by a person who was not directly involved in the data collection to ensure blinding. ## Anthropometric and body composition measurements Body weight was measured using an electronic scale (Filizola PL 50, Filizzola Ltda., Brazil), with a precision of 0.1 kg. Height was measured on a fixed stadiometer with an accuracy of 0.1 cm and a length of 2.20 m. The total body water (TBW), intracellular (ICW) and extracellular water (ECW), fat-free mas (FFM), fat mass (FM), and percentage of fat mass (FM%) were assessed using a spectral bioelectrical impedance analysis and accompanying software (InBody S10, Gangnam-gu, Seoul, Korea). Based on results of a small pilot study (n = 8), the test retest intraclass correlation coefficient (ICC) from our lab were TBW (0.99), ECW (0.99), ICW (0.98), FM (0.97) and FFM (0.99). ## Blood samples and analyses Blood samples (20 ml) were collected at pre-training and after 6 weeks of training at rest and immediately postexercise (within 5 min) during the acute resistance exercise session. The tubes were centrifuged at 3000 rpm for 15 min at 4ºC, and plasma and serum samples were stored at -80ºC until analysis. The Tumor Necrosis Factor-Alpha (TNF-α), soluble TNF-α receptor (sTNFr), IL-6 and IL-10 serum concentrations were determined by enzyme-linked immunosorbent assay (ELISA) technique using high detection sensitivity kits (R&D Sys-tem®, Minneapolis, MN, United States) with range between 15.6 and 1000 pg/mL for TNF-α, 7.8-250 pg/ mL for sTNF-r, 3.13-300 pg/mL for IL-6 and 7.8-500 pg/mL for IL-10; and intra-and inter-assay variation (%) of 4.2-5.2 and 4.6-7.4 (TNF-α), 3.6-5.0 and 3.7-8.8 (sTNF-r), 1.6-4.2 and 3.3-6.4 (IL-6) and 1.7-5.0 and 5.9-7.5 (IL-10), respectively. ## Muscular performance tests and resistance training protocol Prior to all pre-training muscular performance tests, the participants completed two weeks of low-intensity familiarization with the exercises listed in Supplementarywith 2 sets of 15 repetition in each exercise and 60 s of rest. Subjects completed a warm-up before each test, which consisted of 5 min of walking (~6 km.h − 1 ) and a subsequent set of 10 repetitions at approximately 50 % of the 1RM for the first exercise of the specific workout of the day, according to Supplementary. Lower and upper body strength was assessed by 1RM testing in the 45º leg press exercise (1RM-leg press) and the bench press (1RM-bench press) exercises, according the National Strength and Conditioning Association. The acute resistance exercise session consisted of 3 sets of 45º leg press followed by 3 sets of bench press until momentary muscular failure with a load corresponding to 70 % of the 1RM and 90 s of rest between sets and exercise, according to Conrado de Freitas et al.Both peak and mean power output were recorded for each repetition using the Tendo™ Power Output Unit (Tendo Sports Machines, Trencin, Slovak Republic) and the peak power for 3 sets (watts) and mean power for 3 sets (watts) were used. The chronic resistance training program consisted of two progressive phasesand the intensity was controlled by zone of repetition. The sets were executed until concentric muscular failure (i.e. when the participants performed the training with repetitions varying from 10 to 12 RM, they were to execute at least 10 and no more than 12 RM). In the case of the participants executing more repetitions, the load was increased in order to keep within the training zone. Fitness professionals supervised all testing, the exercise routine was conducted face to face (one fitness professional trained two participants), and the total number of repetitions performed was recorded for each set and each exercise, and the total weight lifted was calculated (repetitions × weight lifted × sets). The sum of the total weight lifted for each day was calculated to show total volume per week across the study. # Statistical analysis Using a partial eta-square (0.26) for fat-free mass in the present study, and an alpha value of 5 % via GPower 3.1 software, a 99 % power to our sample size was found by analyzing the difference between groups post hoc. For the outcome measures, an Analysis of covariance (ANCOVA) was applied to adjust for pre-intervention and an Independent t test was used to verify the difference between relative changes (Δ%= post-exercise value minus rest value/rest*100) and confidence interval (CI 95 %) was calculated. For the acute resistance exercise analysis, a mixed between-within subject repeated measures ANOVA was used where the supplementation group (placebo vs. capsaicin) was included as the between-subject factor, exercise effect (rest vs. postexercise) and time (pre-training vs. after 6-weeks of training) were used as the within-subject factors. The inflammatory data was transformed to log, since the data presented non-parametric distribution, according to the Shapiro-Wilk test and a mixed between-within subject ANOVA was used again. The estimated sphericity was verified according to Mauchly's W test and the Greenhouse-Geisser correction was used when necessary. Effect sizes for the ANOVA were calculated using partial eta squared (η 2 ) for time, group and interaction. Data are presented as mean and standard deviation (SD). Statistical significance was set at p < 0.05. The data were analyzed using the Statistical Package for Social Sciences 17.0 (SPSS Inc. Chicago. IL.USA). In addition, it is important highlight that statistician was blind to both groups during data analyses. # Results The Supplementaryshows the total kilocalorie, macronutrient and micronutrient intakes averaged for the three days expressed as grams or relative to body mass at pre-training and post-intervention. There were no statistically significant differences between groups for dietary intake (kcal), carbohydrate, proteins, fats and all micronutrients. Chronic effect of CAP combined with resistance trainingpresents the comparison between Capsiate and placebo groups for body composition and performance pre and post-intervention. For fat-free mass, ANCOVA analysis showed a significant difference between groups (F = 6.610, P = 0.024, η 2 = 0.26). When we analyzed the percentage of changes, there was statistically significant differences between group (t= -2.181, P = 0.043) with a greater increase for CAP (Δ%= 2.1 ± 1.8 %, CI 95 %= 1.2 to 3.4) in relation to placebo (Δ%= 0.7 ± 1.3 %, CI 95 %= -0.2 to 1.6). When we adjusted the FFM effect by the amount of protein consumption, the significant group × time interaction remained (F = 5.725, P = 0.029, η 2 = 0.26). There were no a significant difference between groups for total body mass (F = 0.050, P = 0.825, η 2 = 0.03), fat mass (F = 0.964, P = 0.340, η 2 = 0.05), and percentage of fat mass (F = 0.993, P = 0.333, η 2 = 0.05). There were no significant difference between groups for total body water (F = 1.794, P = 0.198, η 2 = 0.09), extracellular (F = 0.041, P = 0.842, η 2 = 0.002) and intracellular water (F = 0.529, P = 0.477, η 2 = 0.03). For muscle strength in the bench press, ANCOVA analysis showed significant difference between groups (F = 5.283, P = 0.034, η 2 = 0.24). When analyzed the percentage of changes across the intervention, there was statistically significant differences between treatments (t= -2.204, P = 0.041) with greater upper body strength for CAP (Δ%= 13.4 ± 9.1 %) compared to placebo (Δ%= 5.8 ± 5.2 %). There were no significant differences between groups for muscle strength in 45º leg press exercise (F = 0.665, P = 0.426, η 2 = 0.04), power output (F = 18.789, P = 0.001, η 2 = 0.56) and mean peak power (F = 0.054, P = 0.819, η 2 = 0.003) during the acute resistance exercise session. ## Acute resistance exercise session performed at baseline and after 6 weeks of intervention Thepresents the total number of repetition for the acute resistance exercise session in leg press and bench press. For the acute resistance exercise session in leg press, there was a group × exercise (F = 3.779, P = 0.032, η 2 = 0.17) and exercise x time interaction (F = 11.753, P < 0.001, η 2 = 0.39) with a greater number of repetitions performed after 6 weeks of training compared to pretraining and a significant decrease across sets for both groups, however, there were no time × exercise × group interactions (F = 0.002, P = 0.998, η 2 = 0.001). For the acute resistance exercise session in bench press, there was a greater number of repetition after 6 weeks of training compared to baseline (F = 19.737, P < 0.001, η 2 = 0.52) and a significant decrease across sets for both groups (F = 185.601, P < 0.001, η 2 = 0.91), but no time × set × group interaction were observed (F = 0.122, P = 0.886, η 2 = 0.007). presents the changes on inflammatory response after 6 weeks of intervention in the Capsiate and placebo groups. There was a significant exercise × time interaction for TNF-α (F = 4.926, P = 0.040, η 2 = 0.22) with a significant increase post-exercise in relation to rest at preintervention (P = 0.009) but no time × set × group interaction (F = 2.190, P = 0.157, η 2 = 0.11). For sTNF-r, there was a main effect of time (F = 13.449, P = 0.002, η 2 = 0.46) and exercise (F = 28.545, P < 0.001, η 2 = 0.64) but without time × set × group interaction (F = 0.123, P = 0.730, η 2 = 0.008). There was a main effect of exercise for IL-10 (F = 4.964, P = 0.041, η 2 = 0.24) with higher concentration post-exercise compared to rest, however, there was no significant time × set × group interaction (F = 0.197, P = 0.663, η 2 = 0.012). There were not any significant main effects for IL-6 or interactions (F = 0.066, P = 0.801, η 2 = 0.004). # Discussion We hypothesized chronic CAP supplementation would enhance the anti-inflammatory response to resistance training, improve muscular strength and enhance body composition outcomes. While CAP supplementation resulted in greater increases in lean body mass compared to placebo, there were no differences in the immune response to resistance exercise nor performance, with the exception of the bench press 1 RM test. Based on previously reported acute increases in resistance exercise performancewith CAP supplementation, we hypothesized that if supplemented chronically and combined with resistance training, CAP would result in in greater volume accomplished per session, and this would translate to greater muscle hypertrophy. We reject this hypothesis. Although lean body mass increased in CAP, there were no differences between groups for training volume. The interaction between TRVP1 agonists and protein synthesis may explain this discrepancy. By agonizing the TRPV1 receptor, capsaicin increases sarcoplasmic reticulum calcium release and intracellular calcium concentrations and it been shown to increase mTORC1 phosphorylation and activation of downstream targets (p70S6K, S6, Erk1/2 and p38 MAPK) of protein synthesis. In the absence of overload, capsaicin generated muscle hypertrophy in mice was associated with TRPV1mediated activation of mTOR/p70S6K. Additionally, the same authors reported denervated soleus and gastrocnemius muscles atrophied by 32 and 49%, respectively, but CAP alleviated this atrophy. Whether CAP supplementation augments resistance exercise induced increases in mTOR phosphorylation, and whether these increases in mTOR phosphorylation translate to chronic muscle hypertrophy requires further investigation. We also hypothesized CAP would result in greater leg press and bench press 1RM and repetitions to failure volume following 6 weeks of training. We reject this hypothesis as both groups increased strength after 6 weeks of training with no differences between groups. Both groups also increased repetitions to failure following 6 weeks of training in the leg press and bench press. In afferent pain-conducting nerve fibers (nociceptors), chronic capsaicin administration and the resulting calcium influx results in a TRPV1 channel desensitization. Long term in vitro administration of capsaicin has been shown to result in a large reduction and subsequent degradation of both internal and plasma membrane TRPV1 receptors. Additionally, systemic administration of capsaicin to newborn mice resulted in a downregulation of TRPV1 receptors in neuronal but not vascular smooth muscle cells. If a similar down-regulation of TRPV1 receptors occurs in skeletal muscle, it may explain why acute CAP supplementation improved performance in Conrado de Freitas et al., whereas chronic supplementation in the present study was ineffective. In regards to the inflammatory response, TRPV1 activation by both heat (37-42°C) and capsaicin have been shown to increase IL-6 mRNA expression in mouse myoblasts. While the stress induced by exercise results in an anti-inflammatory response and the myokines involved can influence repair and regeneration response in skeletal muscle cells, the dose of CAP used in the present investigation did not potentiate the inflammatory response in untrained men. It is necessary to mentioned that the inflammatory response in our study was measured only immediately post-exercise and at the systemic level, therefore, more research is necessary to investigate cytokine kinetics (i.e. 30 min and 1 h after exercise) and also the paracrine and autocrine signaling with CAP supplementation. A limitation to this study was our inability to sample blood or tissues. Therefore, more research is necessary to investigate the mechanism by which CAP could induce hypertrophy in humans, and to determine if chronic CAP ingestion decreases TRPV1 in skeletal muscle. The form and dose of capsaicin in the present study was well-tolerated and none of the subjects reported gastrointestinal distress. # Conclusions In summary, short term (6 weeks) chronic Capsiate supplementation in conjunction with resistance training increased fat-free mass and upper body strength but did not modulate the inflammatory response to exercise nor performance in young untrained men. The present study can be applied by coaches, trainers and sport nutritionist looking to improve fat-free mass gain and upper body strength during sub-acute chronic capsaicin analog supplementation combined with resistance training.

Reciprocal carbonyl–carbonyl interactions in small molecules and proteins Carbonyl-carbonyl n→π* interactions where a lone pair (n) of the oxygen atom of a carbonyl group is delocalized over the π* orbital of a nearby carbonyl group have attracted a lot of attention in recent years due to their ability to affect the 3D structure of small molecules, polyesters, peptides, and proteins. In this paper, we report the discovery of a "reciprocal" carbonyl-carbonyl interaction with substantial back and forth n→π* and π→π* electron delocalization between neighboring carbonyl groups. We have carried out experimental studies, analyses of crystallographic databases and theoretical calculations to show the presence of this interaction in both small molecules and proteins. In proteins, these interactions are primarily found in polyproline II (PPII) helices. As PPII are the most abundant secondary structures in unfolded proteins, we propose that these local interactions may have implications in protein folding. N ature effectively uses combinations of weak noncovalent interactions in the functional forms of various biologically important molecules such as nucleic acids and proteins [bib_ref] Noncovalent interactions with DNA: An overview, Strekowski [/bib_ref]. Intermolecular noncovalent interactions of varying magnitude are also responsible for the existence of different states of matter. Carbonyl-carbonyl (C═O···C═O) n→π* interactions where one of the lone pairs (n) on the oxygen atom of a carbonyl group is delocalized over the antibonding π* orbital of a nearby carbonyl C═O bond (π* C═O ) along the Bürgi-Dunitz trajectory [bib_ref] Chemical reaction paths. IV. Aspects of O···C=O interactions in crystals, Burgi [/bib_ref] (∠O···C ═ O~109°) have attracted a great deal of attention in recent years [bib_ref] The n→π* interaction: a rapidly emerging non-covalent interaction, Singh [/bib_ref] [bib_ref] Orthogonal multipolar interactions in structural chemistry and biology, Paulini [/bib_ref] [bib_ref] Orthogonal dipolar interactions between amide carbonyl groups, Fischer [/bib_ref] [bib_ref] Nature of amide carbonyl−carbonyl interactions in proteins, Choudhary [/bib_ref] [bib_ref] 13 C NMR reveals no evidence of n−π* interactions in proteins, Worley [/bib_ref] [bib_ref] Intimate interactions with carbonyl groups: dipole-dipole or n→π*?, Kamer [/bib_ref]. Previous studies have shown that C═O···C═O n→π* interactions not only influence geometries of important small molecules [bib_ref] An n→π* Interaction in aspirin: implications for structure and reactivity, Choudhary [/bib_ref] [bib_ref] A Key n→π* interaction in N-acyl homoserine lactones, Newberry [/bib_ref] [bib_ref] n→π* Interactions of amides and thioamides: implications for protein stability, Newberry [/bib_ref] [bib_ref] Collagen stability: insights from NMR spectroscopic and hybrid density functional computational investigations..., Derider [/bib_ref] but also play crucial roles in determining the three dimensional structures of polyesters [bib_ref] n→π* interactions in poly(lactic acid) suggest a role in protein folding, Newberry [/bib_ref] , peptides [bib_ref] Signature of n→π* Interaction in α-helices, Choudhary [/bib_ref] , peptoids [bib_ref] Local and tunable n→π* Interactions regulate amide isomerism in the peptoid backbone, Gorske [/bib_ref] [bib_ref] New strategies for the design of folded peptoids revealed by a survey..., Gorske [/bib_ref] [bib_ref] Extraordinary Robust polyproline type I peptoid helices generated via the incorporation of..., Stringer [/bib_ref] [bib_ref] Cis-Trans Amide Bond Rotamers in β-Peptoids and Peptoids: evaluation of Stereoelectronic Effects..., Laursen [/bib_ref] and proteins [bib_ref] n→π* interactions in proteins, Bartlett [/bib_ref] [bib_ref] The role of Buergi-Dunitz interactions in the structural stability of proteins, Fufezan [/bib_ref] [bib_ref] Interplay of Hydrogen Bonds and n→π* Interactions in Proteins, Bartlett [/bib_ref] [bib_ref] Signatures of n→π* interactions in proteins, Newberry [/bib_ref]. C═O···C═O interactions between the side-chain and backbone carbonyl groups of Asp, Asn, Glu, and Gln were also observed in the high-resolution crystal structures of proteins [bib_ref] Quantum chemical investigations on intraresidue carbonyl−carbonyl contacts in aspartates of high-resolution protein..., Pal [/bib_ref] [bib_ref] Self-contacts in Asx and Glx residues of high-resolution protein structures: role of..., Pal [/bib_ref]. C═O···C═O n→π* interaction is characterized by a short O···C═O distance (d) of less than 3.22 Å [the sum of van der Waals radii of carbon and oxygen atom [bib_ref] van der Waals volumes and radii, Bondi [/bib_ref] ], bond angle ∠O···C═O (θ) of~109°and the pyramidality (Δ, Θ) of the acceptor carbon atom towards the donor oxygen atom [bib_ref] Nature of amide carbonyl−carbonyl interactions in proteins, Choudhary [/bib_ref] [bib_ref] n→π* Interactions of amides and thioamides: implications for protein stability, Newberry [/bib_ref] [bib_ref] Signature of n→π* Interaction in α-helices, Choudhary [/bib_ref] [bib_ref] Signatures of n→π* interactions in proteins, Newberry [/bib_ref] [bib_ref] Pyramidalization of backbone carbonyl carbon atoms in proteins, Esposito [/bib_ref]. Direct spectroscopic evidence for n→π* interaction was recently reported by using gas-phase infrared spectroscopy [bib_ref] Direct spectroscopic evidence for an n→π* interaction, Singh [/bib_ref]. We anticipated that due to n→π* interaction both donor and acceptor C═O bonds will be polarized, which will make the acceptor carbonyl oxygen atom a better electron donor and the donor carbonyl carbon atom a better electron acceptor. The acceptor carbonyl oxygen, therefore, can donate electrons to another nearby carbonyl carbon either to form a sequential chain of O···C contacts [fig_ref] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions [/fig_ref] or it can donate electrons back to the original donor carbonyl carbon atom forming "reciprocal" n→π* interactions [fig_ref] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions [/fig_ref]. Although, the sequential n→π* interactions were previously observed in poly(lactic acid) [bib_ref] n→π* interactions in poly(lactic acid) suggest a role in protein folding, Newberry [/bib_ref] and proteins [bib_ref] n→π* interactions in proteins, Bartlett [/bib_ref] , reciprocal n→π* interactions remained unexplored. Allen and coworkers reported anti-parallel arrangements of carbonyl groups in ketone dimers that were bound together by two intermolecular C═O···C═O short contacts of dipolar nature [bib_ref] Carbonyl-carbonyl interactions can be competitive with hydrogen bonds, Allen [/bib_ref]. Maccallum et al reported a similar geometrical arrangement of carbonyl groups in right-twisted β-strands and observed two chemically distinct dipolar C═O···C═O short contacts [bib_ref] Coulombic attractions between partially charged main-chain atoms stabilise the right-handed twist found..., Maccallum [/bib_ref]. However, these C═O···C═O short contacts were considerably longer than the sum of van der Waals radii of C and O atoms. In this paper, we hypothesized that the polarization of the carbonyl groups by n→π* interactions should lead to back and forth donations between the carbonyl pairs. Based on our hypothesis, we discovered the presence of "reciprocal C═O···C═O interactions" both in small molecules and proteins. To establish the existence of reciprocal C═O···C═O interactions, we designed and synthesized model compounds and carried out X-ray crystallographic and theoretical studies. Further, we carried out Cambridge Structural Database (CSD) [bib_ref] The cambridge structural database in retrospect and prospect, Groom [/bib_ref] and Protein Data Bank (PDB) [bib_ref] The protein data bank, Berman [/bib_ref] analyses to show that these interactions are widely present in small molecules and proteins. In proteins, these interactions are primarily found in random coils and turn regions. Based on our observations we propose that reciprocal C═O···C═O interactions may be a key local interaction that restricts the number of conformers of unfolded proteins and may have a role in protein folding. # Results Reciprocal carbonyl-carbonyl interactions in N,N′-diacylhydrazines. To test our hypothesis of reciprocal n→π* interactions, we have synthesized N,Nʹ-diacylhydrazines 1-8 having various substituents on either side of the carbonyl groups [fig_ref] Figure 2: Model compounds synthesized to study reciprocal n→π* interactions [/fig_ref]. In N,Nʹ-diacylhydrazines 1-8, the two amide carbonyl groups [CO-I and CO-II; [fig_ref] Figure 2: Model compounds synthesized to study reciprocal n→π* interactions [/fig_ref] ] are separated by three covalent bonds and 1,5-type n→π* interactions are feasible from both sides. We propose that due to the repulsion between the nitrogen lone pairs, the N,Nʹ-diacylhydrazines should be nonplanar with the carbonyl groups orientated favorably for reciprocal n→π* interactions. Incorporation of electron donating and withdrawing substituents near the carbonyl groups in 1-8 should help us to tune these interactions. As anticipated, the N,Nʹ-diacylhydrazines [bib_ref] Chemical reaction paths. IV. Aspects of O···C=O interactions in crystals, Burgi [/bib_ref] [bib_ref] The n→π* interaction: a rapidly emerging non-covalent interaction, Singh [/bib_ref] [bib_ref] Orthogonal multipolar interactions in structural chemistry and biology, Paulini [/bib_ref] [bib_ref] Orthogonal dipolar interactions between amide carbonyl groups, Fischer [/bib_ref] crystallized in nonplanar form with the carbonyl groups oriented almost orthogonal to each other (C═O···C═O dihedral angle = +70°to +85°or −70°to −85°) (Supplementary and [fig_ref] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions [/fig_ref]. We observed that in compounds 1-3, that lack any strong electron donating or withdrawing substituent near the carbonyl groups (i.e., X = CH 3 , CH 3 CH 2 ; Y = H, CH 3 ), the two carbonyl groups stay far apart. The crystallographic distances d 1 and d 2 [fig_ref] Figure 2: Model compounds synthesized to study reciprocal n→π* interactions [/fig_ref] in 1-3 are longer than 3.22 Å and natural bond orbital (NBO)calculations carried out on the high-resolution crystal geometries of 1-3 [fig_ref] Table 2 X: -ray crystallographic structural and NBO data of CSD molecules [/fig_ref] show no evidence of n→π* interaction [fig_ref] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the... [/fig_ref]. In compound 4, when X is an electron withdrawing group (e.g., X = CH 2 Cl), an increase in the acceptor ability of the carbonyl CO-II is expected, which should increase n→π* interaction from the oxygen atom of CO-I to the π* C═O orbital of CO-II. However, the inductive electron withdrawal of Cl can be negated by the electron donation from the Cl lone pairs into the antibonding orbitals (σ* and π*) of the adjacent carbonyl group (CO-II). In compound 4, we observed such electron delocalizations from the Cl lone pairs to both σ* and π* orbitals of the C=O bonds of CO-II group, which contributed 0.67 kcal mol −1 to the stabilization. Such electron donation should enhance the donor ability of CO-II in 4. A short crystallographic O 2 ···C 1 distance (d 2 = 3.037 Å is shorter than the sum of van der Waals radii of C and O) and presence of NBO second order perturbation energy [E 2 (n→π*) = 0.34 kcal mol −1 ] for n→π* interaction from CO-II to CO-I in 4 supports this assumption . We anticipate that due to this electron donation from CO-II to CO-I, the CO-I group in 4 will be polarized and the carbonyl oxygen of CO-I will become a better donor. We clearly observed back donation of electrons from CO-I to CO-II in 4 as evidenced by a short crystallographic O 1 ···C 2 distance d 1 of 3.103 Å and n→π* interaction energy [E 1 (n→π*) ] of 0.10 kcal mol −1 obtained by NBO analysis. This is in accordance with our hypothesis that donation from CO-II to CO-I increases the donor ability of CO-I and acceptor ability of CO-II thereby inducing a back donation of electrons from CO-I to CO-II. Similarly, when Y is an electron donating group (e.g., Y = OCH 3 ), CO-I is expected to be a better donor and, accordingly, we observed short distance d 1 in compound 5 . We also observed back donation from CO-II to CO-I in 5 . Among the synthetic compounds 1-8, significantly shorter d 1 and d 2 are observed in 6-8 where electron donating or withdrawing groups are present on both sides of the carbonyl groups. The second order perturbation energies obtained by NBO calculations are also relatively higher for these compounds . The NBO orbital overlaps between the oxygen lone pairs (n O ) and π* C═O orbitals in compound 6 are shown in Figs. 2c, d. Note that in compounds 4 and 6-8 where X = CH 2 Cl or CH 2 Br, d 2 is shorter than d 1 and stronger n→π* interactions from CO-II to CO-I are observed by NBO analysis. This is due to the electron donation from the Cl or Br atom to the σ* C=O and π* C═O orbitals of CO-II, which increases the donor ability of the CO-II oxygen atom. Such electron donations from α-halogens to carbonyl groups and their effect on n→π* interactions were previously reported in the literature [bib_ref] Modulation of an n→π* interaction with α-fluoro groups, Choudhary [/bib_ref] [bib_ref] Conformational analysis of 2-halocyclohexanones: an NMR, theoretical and solvation study, Yoshinaga [/bib_ref]. Interestingly, the values of ∠O···C = O angles θ 1 and θ 2 are much smaller (~82°) in compounds 4-8 where reciprocal n→π* interactions are observed than in 1-3 that lack n→π* interactions . In fact, the values of θ 1 and θ 2 are much smaller than what is expected for one-sided n→π* interactions (∠O···C = O~109°) reported previously [bib_ref] The n→π* interaction: a rapidly emerging non-covalent interaction, Singh [/bib_ref]. This may be due to the geometrical arrangement required for reciprocal n→π* interactions, which forces θ 1 and θ 2 away from the Bürgi-Dunitz trajectory. Another important signature of n→π* interactions is the pyramidality of the acceptor carbonyl carbon atom measured by parameters Δ and Θ [bib_ref] Nature of amide carbonyl−carbonyl interactions in proteins, Choudhary [/bib_ref] [bib_ref] n→π* Interactions of amides and thioamides: implications for protein stability, Newberry [/bib_ref] [bib_ref] Signature of n→π* Interaction in α-helices, Choudhary [/bib_ref] [bib_ref] Signatures of n→π* interactions in proteins, Newberry [/bib_ref] [bib_ref] Pyramidalization of backbone carbonyl carbon atoms in proteins, Esposito [/bib_ref]. Positive values of Δ and Θ indicate pyramidalization of the acceptor carbonyl carbon towards the donor oxygen atom whereas negative values of Δ and Θ indicate pyramidalization of the acceptor carbon away from the donor oxygen atom. In compounds 1-8, however, we have not observed a correlation of pyramidality (Θ) with O···C distance and the strength of n→π* interactions. One reason for this could be the stronger donation from the α-halogen atoms to the nearby carbonyl, which would force the acceptor carbonyl carbons towards the halogen atoms away from the donor oxygen atoms. Also, the crystal packing forces may have some influence in the observed geometries and the pyramidalization of the two nitrogen atoms between the carbonyl groups may influence the pyramidalization of the acceptor carbonyl carbons. Moreover, the individual n→π* interactions in compounds 1-8 may not be strong enough to exert a significant effect on pyramidalization of the carbonyl carbons. Overall, these data suggest that, in compounds 1-8, the geometrical constraints imposed by the repulsion between the nitrogen lone pairs orient the two carbonyl groups favorably for reciprocal n→π* interactions. We could tune these interactions by introducing electron donating or withdrawing substituents near the carbonyl groups. Interestingly, we observed that an increase in n→π* interaction from one side also leads to an increase in the n→π* interaction from the other side in compounds 1-8. This correlation suggests that n→π* interactions in these compounds could be synergistic (Figs. 2e, f). For example, shorter d 1 and higher E 1 (n→π*) values are observed in 4 compared to 3 although 3 and 4 have same the substituent (4-CH 3 -Ph) attached to CO-I. Similarly, higher donation from CO-I to CO-II is observed in 6 compared to 5 although 5 and 6 have same the substituent (4-OCH 3 -Ph) attached to CO-I. [formula] O Y 1, Y = H; X = CH 2 CH 3 X = CH 3 X = CH 3 X = CH 2 CI X = CH 3 X = CH 2 CI X = CH 2 CI X = CH 2 Br 2, Y = H; 3, Y = CH 3 ; 4, Y = CH 3 ; 5, Y = OCH 3 ; 7, Y = Br; 8, Y = OCH 3 ; 6, Y = OCH 3 ; O H N X N H Carbonyl 1 (CO-I) Y d 1 = O 1 ----C 2 d 2 = O 2 ----C 1 N N H H d 1 ,θ 1 d 2 ,θ 2 1 C Carbonyl 2 (CO-II) X 1 O O 2 C 2 θ 1 = ∠ O 1 ----C 2 --- ---O 2 θ 2 = ∠ O 2 ----C 1 --- ---O 1 a b c d 3. [/formula] To find out if geometry optimization has any effect on the computed n→π* interactions in comparison to the unrelaxed X-ray geometries, we also carried out geometry optimizations in compounds 1-8 by freezing the dihedral angles of the side chains involved in reciprocal interactions to their X-ray values and freely optimizing the remaining degrees of freedom (bond lengths, angles, and dihedrals) [fig_ref] Figure 2: Model compounds synthesized to study reciprocal n→π* interactions [/fig_ref]. We observed that reciprocal n→π* interactions were retained after geometry optimizations but they became slightly weaker than what were observed from the NBO calculations on the crystal geometries (Supplementary . The coordinates of the optimized geometries of 1-8 are provided in Supplementary Data 1. We also observed that, during gas phase geometry optimization, in absence of any packing and intermolecular forces that are present in the X-ray geometries, the Cl or Br atoms attached to the methylene carbons in 4, 6-8 moved to an anti-periplanar geometry (trans) with respect to the oxygen atom of the nearby carbonyl group (CO-II). This is probably due to higher hyperconjugative delocalization between the halogen lone pairs and carbonyl π* orbital in the anti-periplanar geometry that would provide more stability to the isolated gas phase molecule. Note that such elongation of carbonyl-carbonyl (O···C) short contacts (weakening of n→π* interactions) in gas phase optimized geometry relative to the X-ray geometries are well known 9, 13, 14 . Reciprocal carbonyl-carbonyl interactions in small organic molecules. To probe whether intramolecular reciprocal C═O···C═O interactions are also present in other small molecules we carried out a CSD search. In our search, we looked for organic molecules having at least two carbonyl groups with intramolecular O 2 ···C 5 (d 1 ) and O 6 ···C 1 (d 2 ) distances ≤ 3.2 Å [fig_ref] Figure 3 X: -ray crystallographic data and NBO overlap diagrams for CSD molecules [/fig_ref]. The search was carried out for cases where the two carbonyl groups are separated by at least three covalent bonds (1,5-type interaction). No restriction was imposed on the ∠O···C = O angles (θ 1 and θ 2 ) during the search. The CSD search provided 1432 molecules which fulfilled our search criteria (Supplementary . The [fig_ref] Table 2 X: -ray crystallographic structural and NBO data of CSD molecules [/fig_ref]. The NBO orbital overlaps between the oxygen lone pairs (n O ) and π* C═O orbitals in one such molecule ( In most of the molecules obtained from the CSD search, reciprocal C═O···C═O interactions were stabilized by both n→π* and π→π* interactions between the carbonyl groups [fig_ref] Table 2 X: -ray crystallographic structural and NBO data of CSD molecules [/fig_ref]. We observed substantial C═O···C═O π→π* interactions in molecules having θ 1 and θ 2 values > 90°( Supplementary . In some cases, π→π* interactions are even stronger than n→π* interactions. When θ 1 and θ 2 values were <90°, π→π* interactions were observed for molecules having relatively shorter O···C distances (both d 1 and d 2 <2.90 Å) and stronger n→π* interactions. We propose that although the contribution of individual orbital interaction is small, the overall contribution of two n→π* and two π→π* interactions to the stabilization of molecules having reciprocal C═O···C═O interactions could be significant. Based on the NBO calculations at B3LYP/6-311 + G(2d,p) level, we observed that reciprocal C═O···C═O interactions contribute 0.11-3.37 kcal mol −1 (with an average value of 0.98 kcal mol −1 ) to the stabilization of small molecules (see the last column in . We observed positive values of Δ and Θ for the carbonyl carbons in most of the molecules from the CSD listed in [fig_ref] Table 2 X: -ray crystallographic structural and NBO data of CSD molecules [/fig_ref] , X-ray crystallographic structural parameters and NBO data for compounds 1-8 [fig_ref] Figure 2: Model compounds synthesized to study reciprocal n→π* interactions [/fig_ref]. E 1 (n→π*) is the NBO second order perturbation energy for electron donation from oxygen lone pair (nO) of the first carbonyl group (CO-I) to the π*C=O orbital of the second carbonyl group (CO-II). E 2 (n→π*) is the NBO second order perturbation energy for electron donation from oxygen lone pair (nO) of the second carbonyl group (CO-II) to the π*C=O orbital of the first carbonyl group (CO-I) [formula] Compounds Y X d 1 d 2 θ 1 θ 2 Δ 1 Δ 2 Θ 1 Θ 2 n→π* (kcal mol −1 ) Total n→π* (kcal mol −1 ) (Å) (Å) (°) (°) (Å) (Å) (°) (°) E 1 (n→π*) E 2 (n→π*) E t (; d2 = O 2 ···C 1 ; θ1 = ∠O 1 ···C 2 = O 2 ; θ2 = ∠O 2 ···C 1 = O 1 (see. E t (n→π*) = E 1 (n→π*) + E 2 (n→π*) [/formula] NP not present ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/s41467-017-00081-x which indicate their pyramidalization towards the donor oxygen atoms. The plots of Θ with O···C distances and the strength of the reciprocal interactions in compounds obtained from the CSD search are shown in [fig_ref] Figure 5: Ramachandran plots and analyses of reciprocal interactions in proteins [/fig_ref]. Although the correlation between pyramidality of second carbonyl (CO-II) carbon (Θ 2 ) and d 1 looks better than the correlation between pyramidality of first carbonyl (CO-I) carbon (Θ 1 ) and d 2 , the CO-I and CO-II are chosen completely randomly in these molecules. As the pyramidalization also depends on other factors like θ and the elasticity of the carbonyl group, a strong correlation between pyramidalization and the O···C distance and strength of n→π* interactions may not be observed in these molecules having different types of carbonyl groups as well as different θ values. To get some insights into the structures of the small molecules having reciprocal C═O···C═O interactions, we manually analyzed small molecules from the CSD having 1,5-type reciprocal interactions with both d 1 and d 2 ≤ 3.00 Å. A total of 249 molecules fulfill the above criteria [1, 5-interaction; both d 1 and d 2 ≤ 3.00 Å]. As can be anticipated, the nature of the two atoms/ groups between the interacting carbonyl groups plays a key role in keeping the two carbonyl groups non coplanar and provides them the conformation required for reciprocal interactions . Interestingly, majority of these molecules (117,~47%) have one heteroatom and one chiral carbon between the two interacting carbonyl pairs, a feature that resembles peptides and proteins. e Chemical structure of a molecule (CCDC reference code: JUHQEK) obtained from the CSD search. The amide carbonyl group is taken as CO-I and the ester carbonyl group is taken as CO-II here. f NBO orbital overlap between oxygen lone pair (n O ) of CO-I and π* C=O orbital of CO-II of JUHQEK. g NBO orbital overlap between oxygen lone pair (n O ) of CO-II and π* C=O orbital of CO-I of JUHQEK. [fig_ref] Figure 3 X: -ray crystallographic data and NBO overlap diagrams for CSD molecules [/fig_ref] Reciprocal carbonyl-carbonyl interactions in proteins. The presence of reciprocal C═O···C═O interactions in the X-ray crystal geometries of small organic molecules inspired us to look for their presence in protein crystal structures. To probe the presence of reciprocal C═O···C═O interactions in proteins, we analyzed a total of 2269 protein crystal structures with resolution ≤ 1.6 Å from the PDB with redundancy (pairwise sequence identity) less than 10%, out of which 2184 showed the presence of reciprocal interactions in them. The PDB protein structures ranked by the number of reciprocal C═O···C═O interactions present in them are included in Supplementary Data 2. For the PDB search, the distance between the carbonyl oxygen of ith amino acid residue and the carbonyl carbon of (i + 1)th amino acid residue is defined as d 1 . [formula] [d 1 = O 2 ···C 5 ; d 2 = O 6 ···C 1 ; θ 1 = ∠O 2 ···C 5 = O 6 ; θ 2 = ∠O 6 ···C 1 = O 2 ( [/formula] The distance between the carbonyl oxygen of (i + 1)th residue and carbonyl carbon of ith residue is defined as d 2 . The corresponding ∠O···C═O angles are defined as θ 1 and θ 2 , respectively (Supplementary . During the search, both d 1 and d 2 were kept ≤ 3.20 Å but no restriction was imposed on θ 1 and θ 2 . The plot of d 1 and d 2 values obtained from the search show that most of them fall in 2.90−3.20 Å range [fig_ref] Figure 4 X|: -ray crystallographic data and NBO overlap diagrams for amino acid pairs [/fig_ref]. The angles θ 1 and θ 2 (~85 ± 15°) deviates significantly from the Bürgi-Dunitz trajectory [fig_ref] Figure 4 X|: -ray crystallographic data and NBO overlap diagrams for amino acid pairs [/fig_ref]. These observations are consistent with the trend that was observed for small molecules discussed above. Analyses of d 1 and d 2 for all amino acid residues in all proteins (2184) studied here show that shorter distances d 1 and d 2 ≤ 3.2 Å fall within the tail of the full distribution [fig_ref] Figure 7: Delocalization energies, charge redistribution and torsion angles [/fig_ref]. In a previous study [bib_ref] n→π* interactions in proteins, Bartlett [/bib_ref] , [fig_ref] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the... [/fig_ref] , [fig_ref] Figure 4 X|: -ray crystallographic data and NBO overlap diagrams for amino acid pairs [/fig_ref]. Similar to CSD molecules, in proteins also we observed substantial C═O···C═O π→π* interactions between the amino acid pairs having θ 1 and θ 2 values > 90°( Supplementary . π→π* NBO orbital overlap between the two carbonyl groups in an amino acid pair is shown in Figs. 4e, f [Leu-Pro (141-142); [PDB: 2x5o]. For molecules having relatively stronger n→π* interactions (both d 1 and d 2 < 2.90 Å), π→π* interactions were observed for θ 1 and θ 2 values <90°also [fig_ref] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the... [/fig_ref]. This indicates that the overall contribution of reciprocal interactions (two n→π* and two π→π* interactions) could be substantial to protein stabilization. Based on the NBO calculations at B3LYP/6-311 + G(2d,p) level, we observed that reciprocal C═O···C═O interactions contribute 0.27-4.41 kcal mol −1 (with an average value of 1.34 kcal mol −1 ) to the stabilization of proteins per amino acid pair (see the last column in . [fig_ref] Figure 3 X: -ray crystallographic data and NBO overlap diagrams for CSD molecules [/fig_ref] , E 1 (n→π*) , E 2 (n→π*) and E t (n→π*) have same meaning as described before in . [CO-I and CO-II are randomly chosen in these molecules] The plot of torsion angles (φ, ψ) of the residue between the two interacting carbonyl groups along with other residues in the proteins show that the reciprocal interactions are mainly concentrated in the polyproline II (PPII), β-turn and right-twisted β-strand regions [fig_ref] Figure 5: Ramachandran plots and analyses of reciprocal interactions in proteins [/fig_ref]. Unlike the one-sided n→π* interactions reported previously [bib_ref] n→π* interactions in proteins, Bartlett [/bib_ref] [bib_ref] The role of Buergi-Dunitz interactions in the structural stability of proteins, Fufezan [/bib_ref] that are abundant in proteins, the abundance of these newly discovered reciprocal C═O···C═O interactions is low (~7.2%). Secondary structure analyses using Stride [bib_ref] Knowledge-based protein secondary structure assignment, Frishman [/bib_ref] show that reciprocal C═O···C═O interactions have considerable abundance in random coils (~20%) and turn regions (10%) of proteins but negligible presence in α-helices (0.35%). This is in contrast to the one-sided n→π* interactions that are most abundant in αhelices [bib_ref] n→π* interactions in proteins, Bartlett [/bib_ref] [bib_ref] The role of Buergi-Dunitz interactions in the structural stability of proteins, Fufezan [/bib_ref]. As PPII helix is not included as an independent secondary structure in most secondary structure predication programs, many PPII helices remain unassigned even though they are present in the experimentally solved structures. We observed that the coil regions having reciprocal C═O···C═O interactions are dominated by PPII structures [(φ, ψ) : (−75°, 145°)]. We have confirmed this by plotting the φ, ψ angles of residues in the random coil regions having reciprocal interactions [fig_ref] Figure 5: Ramachandran plots and analyses of reciprocal interactions in proteins [/fig_ref]. This is not surprising given that PPII conformations are known to dominate coil regions of folded proteins [bib_ref] Polyproline-II helix in proteins: structure and function, Adzhubeil [/bib_ref]. We also manually analyzed 789 reciprocal C═O···C═O interactions in 10 proteins having the highest numbers of reciprocal C═O···C═O interactions coil/PPII and turn regions of these proteins. α-helices that have reciprocal C═O···C═O interactions are distorted, while the β-sheets having reciprocal n→π* interactions are twisted . We also observed reciprocal C═O···C═O interactions between amino acid pairs at the interfaces of different secondary structure types and Supplementary . The other secondary structure that has significant abundance of reciprocal C═O···C═O interactions is β-turn. In a β-turn, the peptide groups (NH and C = O) of the central two amino acids do not participate in any inter-residue hydrogen bonding. Therefore, we assume that these residues may participate in local reciprocal C═O···C═O interactions either between themselves or with their other neighbors, which should compensate for the lack hydrogen bonding interactions in them. A careful examination of the orientations of the carbonyl groups in various common β-turns indicated that reciprocal C═O···C═O interaction may be feasible between the first and the second residues of type Iʹ and II β-turns due to the favorable orientations of the two carbonyl groups but likely to be unfavoured in type I and IIʹ β-turns. In fact, analysis of the 10 protein crystal structures discussed above show that most of the reciprocal C═O···C═O interaction pairs found in β-turns were type II, followed by type IV [fig_ref] Table 2 X: -ray crystallographic structural and NBO data of CSD molecules [/fig_ref]. In 41 cases, the reciprocal n→π* interactions were present between first and the second amino acid residues while in other 43 cases they were between the third and the fourth amino acid residues of β-turns in these 10 proteins. However, in no case the second and the third residues of the βturn were involved in reciprocal C═O···C═O interactions between them . Analysis of distribution of reciprocal C═O···C═O interactions among various amino acids suggests that proline is involved in the largest number of reciprocal C═O···C═O interactions in various proteins followed by glutamic acid and serine [fig_ref] Figure 5: Ramachandran plots and analyses of reciprocal interactions in proteins [/fig_ref]. This trend is different from what was previously observed for one-sided n→π* interactions in α-helices and β-sheets 22 (Pro > Gly > Ala). Analysis of distribution of reciprocal C═O···C═O interactions among the amino acid pairs in various proteins reveals that Pro-Pro is the most abundant pair [fig_ref] Figure 5: Ramachandran plots and analyses of reciprocal interactions in proteins [/fig_ref]. The 10 most prominent amino acid pairs that participate in reciprocal C═O···C═O interactions, all contain a proline residue [fig_ref] Figure 5: Ramachandran plots and analyses of reciprocal interactions in proteins [/fig_ref]. These results may be expected given the abundance of reciprocal interactions in PPII regions. Possible role of reciprocal carbonyl-carbonyl interactions in protein folding. PPII helices and turns are the major secondary structures where reciprocal C═O···C═O interactions are observed. PPII is the major well-defined backbone structure present in denatured, unfolded, and natively unfolded proteins [bib_ref] Conformation of the backbone in unfolded proteins, Shi [/bib_ref] and random coil regions of folded proteins [bib_ref] Polyproline-II helix in proteins: structure and function, Adzhubeil [/bib_ref]. As PPII lacks stable non-local amino acid interactions such as hydrogen bonding, we propose that local reciprocal interactions could possibly contribute to their stability. Levinthal proposed that protein folding is speeded and guided by the rapid formation of local interactions in the unfolded state, which then determine the further folding of the peptide [bib_ref] How to fold graciously, Levinthal [/bib_ref]. The fact that local reciprocal interactions contribute to the stabilization of the PPII conformation that are abundant in unfolded proteins, reciprocal The calculations were carried out at B3LYP/6-311 + G(2d,p) level of theory. For the definitions of d 1 , d 2 , θ 1 and θ 2 see . E 1 (n→π*) , E 2 (n→π*) and E t (n→π*) have same meaning as described before in . [CO-I and CO-II are randomly chosen in these molecules] NP not present interactions could play a role in protein folding. Also, turn regions that are stabilized by reciprocal interactions are known to act as nucleation sites for protein folding. Therefore, an open question is how important such reciprocal interactions might be for protein folding. Nature of reciprocal carbonyl-carbonyl interactions. The nature of C═O···C═O interactions has been debated in the literature. While some consider them n→π* orbital interactions 9, 11 , others believe them to be dipolar in nature [bib_ref] Orthogonal multipolar interactions in structural chemistry and biology, Paulini [/bib_ref] [bib_ref] Orthogonal dipolar interactions between amide carbonyl groups, Fischer [/bib_ref] [bib_ref] 13 C NMR reveals no evidence of n−π* interactions in proteins, Worley [/bib_ref]. We have so far discussed reciprocal C═O···C═O interactions as n→π* and π→π* orbital interactions because of the following reasons. Firstly, the plots of the n→π* and sum of n→π* and π→π* orbital interaction energies against the O···C distances (d) show a strong correlation [fig_ref] Figure 7: Delocalization energies, charge redistribution and torsion angles [/fig_ref]. In [fig_ref] Figure 7: Delocalization energies, charge redistribution and torsion angles [/fig_ref] , we have plotted the distances (d 1 and d 2 values) against the stabilization energies due to n→π* interactions [NBO second order perturbation energies E 1 (n→π*) and E 2 (n→π*) ] reported in Tables 1-3. The plot suggests that the stabilization energies E (n→π*) for n→π* interactions decreases with an increase in the O···C (d) in synthetic molecules 1-8, molecules taken from CSD and interacting amino acid pairs obtained from PDB [fig_ref] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions [/fig_ref]. The overall orbital interaction energies (sum of n→π* [E (n→π*) ] and π→π* [E (π→π*) ] interaction energies reported in Tables 1-3) plotted in [fig_ref] Figure 7: Delocalization energies, charge redistribution and torsion angles [/fig_ref] also show a similar correlation with O···C (d) distances. These correlations indicate that orbital interaction is the major mechanism for the stabilization of these reciprocal C═O···C═O short contacts. Secondly, we carried out NBO deletion analysis on all the molecules reported in [fig_ref] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the... [/fig_ref] and observed that deletion of n→π* interactions increases charge on donor oxygen lone pair (n O ) and depletes it on acceptor carbonyl π* C═O orbital, which correlate well with the strength of O···C distances [fig_ref] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions [/fig_ref]. Similarly, deletion of π→π* interactions increases charge on π C═O orbital of donor carbonyl and depletes it on π* C═O orbital of the acceptor carbonyl, which also can be correlated to the strength of C═O···C═O short contacts [fig_ref] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions [/fig_ref]. The overall accumulation of We conclude that reciprocal carbonyl-carbonyl interactions exist both in small organic molecules and proteins. However, due to geometrical constraints associated with such interactions, the approach of the donor oxygen atoms to the acceptor carbon atoms deviates significantly from the Bürgi-Dunitz trajectory, and therefore, electron delocalization between the oxygen lone pair (n O ) and π* C═O orbital is weak. This weak donation from the first carbonyl group to the second is compensated by a back donation from the second carbonyl group to the first. In many cases, reciprocal π→π* interactions were also observed along with reciprocal n→π* interactions and their overall contributions to the stabilization of molecules having reciprocal C═O···C═O short contacts could be significant. In proteins, C═O···C═O n→π* interactions are present in all types of secondary structures. While one-sided n→π* interactions are prevalent in α-helices 22, 23 , reciprocal interactions are abundant in PPII helices and turn regions. Prevalence of reciprocal C═O···C═O interactions in PPII helices and turn regions of proteins suggests a possible role for these interactions in protein folding. Further, the presence of reciprocal C═O···C═O interactions in distorted α-helices and twisted β-sheets suggests that these interactions could stabilize secondary structures that deviate from their regular geometries. The reciprocal C═O···C═O interactions present at the interface of two different types of secondary structures could also help in stabilizing the strained amino acid residues that are present at these interfaces. In future, it would be interesting to investigate the ability of amino acid pairs having high propensity to get involved in reciprocal C═O···C═O interactions to stabilize PPII a b d c e Reciprocal carbonyl-carbonyl interactions in various secondary structures. a PPII-helix; b β-turn; c Right-twisted β-strand; d α-helix; e interface of αhelix and β-sheet. The Figures are generated by using PyMOL helices and β-turns. It would also be interesting to investigate if some non-peptidic fragments obtained from the CSD search having strong reciprocal C═O···C═O interactions could be used to stabilize PPII conformation or design peptide-turns. Finally, an energy decomposition analysis would provide better understanding of the forces that contributes to the stabilization of reciprocal C═O···C═O interactions. # Methods Crystallization method. Single crystals of compounds 1-8 were grown by slow evaporation. Various solvent combinations were used to crystallize the compounds either at room temperature or low temperature (4°C). Details of the crystallization conditions are given in . X-ray crystal structure determination method. Single crystal structures of compound 1-8 were determined by measuring X-ray intensity data. Bruker D8Venture APEX 3 42 single crystal home source X-ray diffractometer equipped with CMOS PHOTON 100 detector and Monochromated microfocus sources Mo Kα radiation (λ = 0.71073 Å) were used for data collection in phi (ϕ) and omega (ω) scan strategy at room temperature (298 K). The data was processed using SAINTand absorption correction was done using SADABS 44 implemented in APEX 3. For structure solution XSHELL program based on SHELX [bib_ref] A short history of SHELX, Sheldrick [/bib_ref] was used. The non-hydrogen atoms were refined anisotropically and located in successive difference Fourier syntheses. The hydrogen atoms were fixed to neutron bond length using appropriate HFIX commands. ORTEP diagrams of compounds 1-8 (CCDC 1486577-1486584) is provided in [fig_ref] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions [/fig_ref]. Compound 5 crystallized with a water molecule in the asymmetric unit. However, for clarity we have not shown the water molecule in its ORTEP diagram. Compound 7 has disorder at chlorine atom; the occupancy of disordered chlorine atom namely Cl1A [fig_ref] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the... [/fig_ref]. When the x-axis is d 1 , E 1 (n→π*) is plotted in the y-axis and when the x-axis is d 2 , E 2 (n→π*) is plotted in the y-axis. The d 1 , d 2 , E 1 (n→π*) and E 2 (n→π*) values are taken from Tables 1-3. The n→π* interaction energies were computed at B3LYP/6-311 + G(2d,p) level of theory. b Plot of overall orbital interaction energy (sum of n→π* and π→π* interaction energies) between the interacting carbonyl pairs against crystallographic O···C distances (d 1 and d 2 ) in molecules shown in [fig_ref] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the... [/fig_ref]. When the x-axis is d 1 , E 1 (n→π*) + E 1 (π→π*) is plotted in the y-axis and when the x-axis is d 2 , E 2 (n→π*) + E 2 (π→π*) is plotted in the y-axis. d 1 , d 2 E 1 (n→π*) and E 2 (n→π*) , values are taken from Tables 1-3. E 1 (π→π*) and E 2 (π→π*) values are taken from Supplementary [fig_ref] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the... [/fig_ref]. The orbital interaction energies were computed at B3LYP/6-311 + G(2d,p) level of theory. c Plot of accumulation of charges on the π* C=O orbital of CO-II due to donation from lone pairs of oxygen and π C=O orbital of CO-I against d 1 . d Plot of accumulation of charges on the π* C=O orbital of CO-I due to donation from lone pairs of oxygen and π C=O orbital of CO-II against d 2 . The solid curves in a-d are drawn for convenience. e Histogram plot showing the frequency of the C 1 ═O 2 ···C 5 ═O 6 dihedral angles (see [fig_ref] Figure 3 X: -ray crystallographic data and NBO overlap diagrams for CSD molecules [/fig_ref] for atom numbers) for 1432 molecules obtained from the CSD search. f Histogram plot showing the frequency of the C 5 ═O 6 ···C 1 ═O 2 dihedral angles (see [fig_ref] Figure 3 X: -ray crystallographic data and NBO overlap diagrams for CSD molecules [/fig_ref] for atom numbers) for 1432 molecules obtained from the CSD search and Cl1B was refined using the PART command. Similar ADP restraint SIMUand rigid bond restraint DELU 46 was applied to stabilize the anisotropic refinement. SADIinstruction was used to restrain the distance to equal. The anisotropic displacement parameter for disordered chlorine atom was fixed using EADPconstraint. CSD analysis. Intramolecular C═O···C═O noncovalent interactions were searched and structural data were retrieved from Cambridge Structural Database 33 (CSD version 5.21 Nov. 2015) using Conquest 47 (version 1.18) program. The fragment chosen for the search is shown in [fig_ref] Figure 3 X: -ray crystallographic data and NBO overlap diagrams for CSD molecules [/fig_ref] , where X is indicative for any atom. Only unique matching fragments were taken and the fragment was chosen in such a way that there are at least two carbonyl groups irrespective of their nature. Distances d 1 (O 2 -C 5 ) and d 2 (O 6 -C 1 ) are restricted to ≤3.2 Å. Angles [O 2 -C 5 -O 6 (θ 1 ) and O 6 -C 1 -O 2 (θ 2 )] and dihedral angles (C 1 -O 2 -C 5 -O 6 and C 5 -O 6 -C 1 -O 2 ) were printed without any restriction. Only crystalline, non-ionic and non-polymeric organic molecules having no disorder and error with R factor ≤ 5% having at least three covalent bond separations between the carbonyl groups were considered in this search. PDB analysis. A subset of 2269 protein was culled out from RCSB PDB 34 using a search criterion of resolution <1.6 Å with redundancy (pairwise sequence identity) less than 10%, downloaded on 19 January 2016. Out of 2269 proteins, 2184 showed the reciprocal n→π* interaction. For proteins existing in polymeric form or for proteins containing amino acids in more than one conformation, Chain A and conformation A were chosen, except for 57 proteins where chain A is absent. Distance d 1 is defined as distance between the i th amide oxygen to the subsequent (i + 1) th amide carbon, while d 2 is defined as distance between the (i + 1) th amide oxygen to the i th amide carbon . We used d 1 ≤ 3.2 Å and d 2 ≤ 3.2 Å criteria for selecting amino acid pairs participating in reciprocal n→π* interactions. Secondary structure assignment was done using the Stride code [bib_ref] Polyproline-II helix in proteins: structure and function, Adzhubeil [/bib_ref]. Ramachandran plots were generated for the proteins using Gnuplot (http://www.gnuplot.info/). Computational methods. All the calculations were performed by using Gaussian09 suite of quantum chemistry programs. The Hartree-Fock (HF) [bib_ref] A simplification of the Hartree-Fock method, Slater [/bib_ref] and the hybrid Becke 3-Lee-Yang-Parr (B3LYP) 50, 51 exchange correlation functional with 6-311 + G (2d,p) basis set were used for the calculations. Natural bond orbital (NBO)analyses were performed on the crystal geometries of the synthetic molecules and small organic molecules obtained from CSD search. For proteins, the coordinates of the interacting amino acid residue pair were extracted using PyMOL. The α-carbons of the amino acid residues adjacent to N and C termini of the amino acid pair were also included, so as to mimic a dipeptide with N and C termini capped with N(CO)Me and (CO)NMe, respectively. Finally, hydrogen atoms were added to the structure using PyMOL . NBO analyses were carried out on crystal geometries at B3LYP/6-311 + G(2d,p) and HF/6-311 + G(2d,p) level of theory. The NBO second order perturbative energies E (n→π*) and E (π→π*) obtained from NBO calculations were taken as the stabilization energy due to n→π* and π→π* interactions. NBO deletion analysis was carried out on crystal geometries at HF/6-311 + G(2d,p) level of theory. Data availability. The authors declare that the data supporting the findings of this study are available within the paper and its Supplementary Information files, and also are available from the corresponding author upon reasonable request. X-ray crystallographic data for structures reported in this study have been deposited at the Cambridge Crystallographic Data Centre (CCDC), under deposition number CCDC 1486577-1486584. These data can be obtained free of charge from the CCDC via www.ccdc.cam.ac.uk/. Received: 26 July 2016 Accepted: 31 May 2017 [fig] Figure 1: Schematic illustration of a one-sided and b reciprocal n→π* interactions. Curved dotted arrows indicate n→π* interactions [/fig] [fig] Figure 2: Model compounds synthesized to study reciprocal n→π* interactions. a Chemical structures of N,Nʹ-diacylhydrazines (1-8). b Definition of different structural parameters in N,Nʹ-diacylhydrazines 1-8; d 1 = O 1 ···C 2 ; d 2 = O 2 ···C 1 ; θ 1 = ∠O 1 ···C 2 = O 2 ; θ 2 = ∠O 2 ···C 1 = O 1 . c NBO orbital overlap between oxygen lone pair (n O ) of CO-I and π* C=O orbital of CO-II of compound 6. d NBO orbital overlap between oxygen lone pair (n O ) of CO-II and π* C=O orbital of CO-I of compound 6. e Plot showing correlation between O···C distances (d 1 and d 2 ) in compounds 1-8 [Linear fitting; Pearson correlation coefficient = 0.9906]. f Plot showing correlation between reciprocal n→π* interaction energies [E 1 (n→π*) and E 2 (n→π*) ] in compounds 1-8 [Linear fitting; Pearson correlation coefficient = 0.938]. Curved dotted arrows indicate n→π* interactions [/fig] [fig] Figure 3 X: -ray crystallographic data and NBO overlap diagrams for CSD molecules. a Plot showing the distribution of O···C distances (d 1 and d 2 ) in molecules obtained from the CSD search. b Plot showing the distribution of ∠O···C = O angles (θ 1 and θ 2 ) in molecules obtained from the CSD search. c Plot of distance d 1 vs. angle θ 1 in molecules obtained from the CSD search. d Plot of distance d 2 vs. angle θ 2 in molecules obtained from the CSD search. [/fig] [fig] Figure 4 X|: -ray crystallographic data and NBO overlap diagrams for amino acid pairs. a Plot showing the distribution of O···C distances (d 1 and d 2 ) in amino acid pairs in proteins having reciprocal C═O···C═O interactions. b Plot showing the distribution of ∠O···C = O angles θ 1 and θ 2 in amino acid pairs in proteins having reciprocal C═O···C═O interactions. The vertical and horizontal blue lines are drawn at θ 1 = 99°and θ 2 = 99°, respectively. c NBO orbital overlap between oxygen lone pair (n O ) of CO-I and π* C=O orbital of CO-II of Leu-Pro (141-142) [PDB: 2x5o]. d NBO orbital overlap between oxygen lone pair (n O ) of CO-II and π* C=O orbital of CO-I of Leu-Pro (141-142) [PDB: 2 × 5o]. e NBO orbital overlap between the π orbital of C = O bond of CO-I and π* C=O orbital of CO-II of Leu-Pro (141-142) [PDB: 2x5o]. f NBO orbital overlap between the π orbital of C = O bond of CO-II and π* C=O orbital of CO-I of Leu-Pro (141-142) [DOI: 10.1038/s41467-017-00081-x | www.nature.com/naturecommunications [/fig] [fig] Figure 5: Ramachandran plots and analyses of reciprocal interactions in proteins. a Ramachandran plot generated by plotting torsion angles (φ, ψ) of all residues in 2184 protein structures (blue) and torsion angles (φ, ψ) of the residue between the two interacting carbonyl groups involved in reciprocal C═O···C═O interactions (yellow). b Ramachandran plot generated by plotting torsion angles (φ, ψ) of all residues in 2184 protein structures (blue) and torsion angles (φ, ψ) of the residue between the two interacting carbonyl groups involved in reciprocal C═O···C═O interactions present only in the coil regions (yellow). c Plot showing percentage distribution of amino acids involved in reciprocal C═O···C═O interactions. d Plot showing percentage distribution of amino acid pairs involved in reciprocal C═O···C═O interactions [/fig] [fig] Figure 7: Delocalization energies, charge redistribution and torsion angles. a Plot of n→π* interaction energies between the interacting carbonyl pairs against crystallographic O···C distances (d 1 and d 2 ) in molecules shown in [/fig] [table] Table 2 X: -ray crystallographic structural and NBO data of CSD molecules [/table] [table] Table 3 X: -ray crystallographic structural and NBO data for amino acid pairs from the PDB [/table]

Comparison of biodegradable and durable polymer drug-eluting stents in acute coronary syndrome: a meta-analysis ⇒ This meta-analysis included randomised controlled trials with long-term follow-ups. ⇒ The large sample size ensures adequate statistical power to detect even a small effect of interest. ⇒ Heterogeneity among the biodegradable polymer drug-eluting stents may distort the reported results. ⇒ The differences in the duration of dual antiplatelet therapy may influence clinical outcomes. abStraCt objective To compare the safety and effectiveness between biodegradable polymer drug-eluting stents (BP-DES) and durable polymer drug-eluting stents (DP-DES) in patients with acute coronary syndrome (ACS). design Meta-analysis of randomised controlled trials (RCTs). Primary and secondary outcome measures Major adverse cardiovascular events (MACEs) were considered the primary endpoint. Efficacy endpoints included target vessel revascularisation (TVR) and target lesion revascularisation (TLR). Safety endpoints included allcause death, cardiac death, target vessel myocardial infarction and stent thrombosis (ST). methods We searched PubMed, Medline, Embase and the Cochrane Controlled Register of Trials for comparative studies of BP-DES and DP-DES in patients with ACS from January 2000 to July 2021. Statistical pooling was performed to estimate incidence using a random-effects model with generic inverse-variance weighting. Risk estimates were computed with 95% CIs. results Eight articles with seven RCTs that compared BP-DES and DP-DES in patients with ACS were identified and included in the qualitative and quantitative analyses. There was no difference in the baseline characteristics, except for the number of smoking patients (OR: 1.13, 95% CI 1.03 to 1.24; p=0.008, I 2 =29%), which was significantly lower in the BP-DES group. The metaanalysis demonstrated that MACEs, efficacy endpoints and safety endpoints were similar between the groups at 1 year. However, the incidence of total ST was significantly different between the BP-DES and DP-DES groups in the follow-up period (p=0.0001). Further analysis showed a statistically significant difference in MACEs (OR: 0.71, 95% CI 0.57 to 0.88; p=0.002, I 2 =0 %), TLR (OR: 0.71, 95% CI 0.51 to 1.01; p=0.05, I 2 =0%), TVR (OR: 0.70, 95% CI 0.52 to 0.94; p=0.002, I 2 =15%), total ST incidence (OR: 0.59, 95% CI 0.46 to 0.77; p=0.0001, I 2 =48%) and ST incidence (OR: 0.63, 95% CI 0.47 to 0.85; p=0.002, I 2 =0%) over 2 years. Conclusion This meta-analysis revealed that both stent types demonstrated excellent safety and efficacy profiles at 12 months. However, a slight increase in MACEs, TLR, TVR and ST incidence was observed in the DP-DES group over the 2-year follow-up period, suggesting that BP-DES may be more favourable when treating patients with ACS. trial registration number NCT00389220.introduCtion Percutaneous coronary intervention (PCI) is the current standard of care for patients with coronary artery disease, particularly acute coronary syndrome (ACS). 1 2 Unlike baremetal stents (BMS), drug-eluting stents (DES) use antiproliferative agents embedded in a polymer coating on the stent's surface, which inhibit neointimal hyperplasia to reduce the risk of restenosis. 3 DES have substantially improved clinical outcomes; however, the first-generation durable polymer DES (DP-DES) were known to release sirolimus or paclitaxel, and were associated with similar risks of death and myocardial infarction compared with those of BMS beyond 1 year after implantation. 4 Later, the second-generation DP-DES were confirmed to have lower restenosis rates than the first-generation devices and demonstrated reduced rates of stent thrombosis (ST). 5 Recently, very late ST and neoatherosclerosis, with adverse clinical outcomes, have been observed with the second-generation DP-DES, which has improved the biocompatibility of the polymer. 6 Late stent failure has been attributed to delayed endothelial healing secondary to a hypersensitivity reaction due to the DP. 7 To address this potential limitation of DP-DES, biodegradable polymer DES (BP-DES) have been developed. Theoretically, BP-DES would lead to a reduction in vascular inflammation and a decreased risk of late abStraCt objective To compare the safety and effectiveness between biodegradable polymer drug-eluting stents (BP-DES) and durable polymer drug-eluting stents (DP-DES) in patients with acute coronary syndrome (ACS). design Meta-analysis of randomised controlled trials (RCTs). Primary and secondary outcome measures Major adverse cardiovascular events (MACEs) were considered the primary endpoint. Efficacy endpoints included target vessel revascularisation (TVR) and target lesion revascularisation (TLR). Safety endpoints included allcause death, cardiac death, target vessel myocardial infarction and stent thrombosis (ST). methods We searched PubMed, Medline, Embase and the Cochrane Controlled Register of Trials for comparative studies of BP-DES and DP-DES in patients with ACS from January 2000 to July 2021. Statistical pooling was performed to estimate incidence using a random-effects model with generic inverse-variance weighting. Risk estimates were computed with 95% CIs. results Eight articles with seven RCTs that compared BP-DES and DP-DES in patients with ACS were identified and included in the qualitative and quantitative analyses. There was no difference in the baseline characteristics, except for the number of smoking patients (OR: 1.13, 95% CI 1.03 to 1.24; p=0.008, I 2 =29%), which was significantly lower in the BP-DES group. The metaanalysis demonstrated that MACEs, efficacy endpoints and safety endpoints were similar between the groups at 1 year. However, the incidence of total ST was significantly different between the BP-DES and DP-DES groups in the follow-up period (p=0.0001). Further analysis showed a statistically significant difference in MACEs (OR: 0.71, 95% CI 0.57 to 0.88; p=0.002, I 2 =0 %), TLR (OR: 0.71, 95% CI 0.51 to 1.01; p=0.05, I 2 =0%), TVR (OR: 0.70, 95% CI 0.52 to 0.94; p=0.002, I 2 =15%), total ST incidence (OR: 0.59, 95% CI 0.46 to 0.77; p=0.0001, I 2 =48%) and ST incidence (OR: 0.63, 95% CI 0.47 to 0.85; p=0.002, I 2 =0%) over 2 years. Conclusion This meta-analysis revealed that both stent types demonstrated excellent safety and efficacy profiles at 12 months. However, a slight increase in MACEs, TLR, TVR and ST incidence was observed in the DP-DES group over the 2-year follow-up period, suggesting that BP-DES may be more favourable when treating patients with ACS. trial registration number NCT00389220. introduCtion Percutaneous coronary intervention (PCI) is the current standard of care for patients with coronary artery disease, particularly acute coronary syndrome (ACS). Unlike baremetal stents (BMS), drug-eluting stents (DES) use antiproliferative agents embedded in a polymer coating on the stent's surface, which inhibit neointimal hyperplasia to reduce the risk of restenosis. 3 DES have substantially improved clinical outcomes; however, the first-generation durable polymer DES (DP-DES) were known to release sirolimus or paclitaxel, and were associated with similar risks of death and myocardial infarction compared with those of BMS beyond 1 year after implantation. [bib_ref] Comparison of angioplasty with infusion of tirofiban or abciximab and with implantation..., Valgimigli [/bib_ref] Later, the second-generation DP-DES were confirmed to have lower restenosis rates than the first-generation devices and demonstrated reduced rates of stent thrombosis (ST). [bib_ref] Very late coronary stent thrombosis of a newer-generation everolimus-eluting stent compared with..., Räber [/bib_ref] Recently, very late ST and neoatherosclerosis, with adverse clinical outcomes, have been observed with the second-generation DP-DES, which has improved the biocompatibility of the polymer. [bib_ref] Meta-Analysis of randomized clinical trials comparing biodegradable polymer drug-eluting stent to second-generation..., El-Hayek [/bib_ref] Late stent failure has been attributed to delayed endothelial healing secondary to a hypersensitivity reaction due to the DP. [bib_ref] Temporal course of neointimal formation after drug-eluting stent placement: is our understanding..., Finn [/bib_ref] To address this potential limitation of DP-DES, biodegradable polymer DES (BP-DES) have been developed. Theoretically, BP-DES would lead to a reduction in vascular inflammation and a decreased risk of late Open access stent-related complications due to the advantage of leaving the BMS only after complete drug elution and polymer degradation. BP-DES have been observed to reduce the rate of major adverse cardiac events (MACEs) compared with BMS 8 and first-generation DP-DES. [bib_ref] Everolimus-Eluting stent versus bare-metal stent in ST-segment elevation myocardial infarction (examination): 1..., Sabate [/bib_ref] Studies of patients who underwent PCI revealed that the devicerelated outcomes were comparable between BP-DES and second-generation DP-DES. [bib_ref] Very thin strut biodegradable polymer everolimus-eluting and sirolimus-eluting stents versus durable polymer..., Von Birgelen [/bib_ref] [bib_ref] Ultrathin strut biodegradable polymer sirolimus-eluting stent versus durable polymer everolimuseluting stent for..., Pilgrim [/bib_ref] [bib_ref] Abluminal biodegradable polymer biolimus-eluting stent versus durable polymer everolimus-eluting stent (compare II):..., Smits [/bib_ref] [bib_ref] Biodegradable polymer biolimus-eluting stent versus durable polymer everolimus-eluting stent: a randomized, controlled,..., Natsuaki [/bib_ref] Thus, BP-DES would be expected to reduce the risk of ST-related MACEs beyond the first year compared with that of DP-DES. However, previous studies enrolled a significant proportion of stable angina patients. ACS confers an increased risk of adverse outcomes due to plaque characteristics, including culprit lesions, thrombus burden and persistent inflammation, compared with stable coronary artery diseases. ACS also increases the risk of delayed arterial healing and vessel remodelling, 14 reflected by higher rates of incomplete stent strut coverage and malpositioning. [bib_ref] Correlation of intravascular ultrasound findings with histopathological analysis of thrombus aspirates in..., Cook [/bib_ref] Recently, many randomised trials have been performed to compare the efficacy and safety of DP-DES and BP-DES in patients with ACS who underwent PCI. In this metaanalysis, we aimed to summarise the studies comparing the two polymer technologies in patients with ACS and analyse the safety and effectiveness of these therapeutic options. # Methods ## Search strategy and registration This study was conducted according to the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines and was approved by the institutional review board of the Second Xiangya Hospital, Central South University. The protocol was registered with PROSPERO (CRD42021253412). Based on the PRISMA statement, PubMed, Medline, Embase and the Cochrane Controlled Register of Trials databases were searched for comparative studies of BP-DES and DP-DES that were used in the treatment of patients with ACS who underwent PCI. The following search terms were used: "BP-DES," "biodegradable," "bioabsorbable," "bioabsorbable polymer drug-eluting stent," "biodegradable polymer drug-eluting stent," "DP-DES," "durable polymer," "durable polymer drug-eluting stent," "acute coronary syndrome," "ACS," "AMI," "Acute myocardial infarction," "Non ST segment elevation myocardial infarction," "ST segment elevation myocardial infarction," "NSTEMI," and "STEMI." We also reviewed prior meta-analyses and the reference lists of the original trials and reviewed articles to identify further studies. Only English language articles published in peer-reviewed journals from January 2000 to July 2021 were selected. Analyses were conducted by two independent reviewers. ## Eligibility criteria The inclusion criteria for this meta-analysis were as follows: (1) randomised controlled trials (RCTs) comparing BP-DES and DP-DES in the treatment of patients with ACS who underwent PCI; (2) data reporting patients' baseline characteristics, follow-up durations, outcomes at the primary, safety and efficacy endpoints; (3) mean follow-up time over 12 months; and (4) full-text articles. The exclusion criteria for the meta-analysis were as follows: (1) duplications of samples and reports (evaluated by two independent reviewers); (2) case reports/ series; and (3) studies involving data from a national database. data extraction and outcome measurement Two authors (HY and TW) systematically screened the titles and abstracts of publications retrieved using the search strategy to select studies that met the above inclusion criteria. Any disagreement regarding the eligibility of particular studies was resolved through discussion and involvement of a third author (ZW), when necessary. First, baseline characteristics, including the name Open access of the first author, year of publication, study design, country of origin, number of patients, mean age of participants and mean duration of follow-up, were gathered from each included article. In addition, data on sex; body mass index; the presence of hypertension, diabetes, dyslipidaemia, chronic kidney disease, peripheral vessel disease or smoking; left ventricular ejection fraction (LVEF); number of stents per person; and total stent length were collected for evaluation of procedurerelated risks. MACEs were considered the primary endpoint. The efficacy endpoints included target vessel revascularisation (TVR) and target lesion revascularisation (TLR). In addition, all-cause death, cardiac death, target vessel myocardial infarction (TVMI), and ST were used as endpoints to evaluate the safety of BP-DES and DP-DES. The Risk of Bias 2 tool was used to assess the quality of RCTs based on sequence generation; randomised group allocation; concealment; blinding of participants, personnel and outcome assessors; incomplete data; selectivity; outcome reporting; and other sources of bias (online supplementary material 1). [bib_ref] ROBINS-I: a tool for assessing risk of bias in non-randomised studies of..., Sterne [/bib_ref] data analysis and synthesis Continuous variables were reported as the mean (SD), and categorical variables were expressed as numbers. Statistical pooling was performed to estimate incidence, according to a random-effects model with generic inversevariance weighting. We computed risk estimates with 95% CIs, using RevMan V.5.3 (The Cochrane Collaboration, The NordicCochrane Centre, Copenhagen, Denmark). Hypothesis testing for superiority was set at the two-tailed 0.05 level. Hypothesis testing for statistical homogeneity was set at the two-tailed 0.10 level and was based on the Cochran Q test, with I 2 values of 25%, 50% and 75% representing mild, moderate and severe heterogeneity, respectively. # Results # Search results A total of 895 articles, written in English, were identified through the literature search. After an initial screening of the titles and abstracts, 803 articles were eliminated, as they were not related to the topic of this study. Following implanted with BP-DES or DP-DES. The meta-analysis demonstrated that the number of smoking patients (OR: 1.13, 95% CI 1.03 to 1.24; p=0.008, I 2 =29%) was significantly lower in the BP-DES group than that in the DP-DES group (figures 1-3). Primary endpoint: maCes reported during follow-up periods of 1-5 years, 1 year and over 2 years MACEs, including all-cause death, recurrent myocardial infarction (MI), or any coronary repeat revascularisation involving TLR, TVR and non-TVR, were considered to be the primary endpoint of the trials. A meta-analysis indicated no statistically significant difference in the MACEs in a follow-up period ranging from 1 to 5 years between the two groups (OR: 0.87, 95% CI 0.75 to 1.01; p=0.07, I 2 =50%). Of the five studies that published 1-year outcomes, MACEs were not significantly different between the BP-DES and DP-DES groups (OR: 0.97, 95% CI 0.81 to 1.16; p=0.74, I 2 =44%). However, MACEs with follow-up periods of over 2 years were significantly lower in the BP-DES group (OR: 0.71, 95% CI 0.57 to 0.88; p=0.002, I 2 =0 %) (figure 4). efficacy endpoint: tVr and tlr reported during follow-up periods of 1-5 years, 1 year and over 2 years TLR and TVR were considered the efficacy endpoints of the trials. The meta-analysis indicated no statistically significant difference in TLR in the follow-up periods ranging from 1 to 5 years between the two groups (OR: 0.78, 95% CI 0.61 to 1.00; p=0.05, I 2 =48%). Among the five studies that published 1-year data, TLR was not significantly different between the BP-DES and DP-DES groups (OR: 0.72, 95% CI 0.40 to 1.31; p=0.29, I 2 =65%). The metaanalysis indicated no statistically significant difference in TVR in the follow-up periods ranging from 1 to 5 years (OR: 1.01, 95% CI 0.79 to 1.28; p=0.96, I 2 =46%) or in the three publications with 1-year follow-up periods (OR: 0.98, 95% CI 0.40 to 2.38; p=0.96, I 2 =76%). However, the difference in TLR was statistically significant in four RCT studies with follow-up periods of over 2 years (OR: 0.71, 95% CI 0.51 to 1.01; p=0.05, I 2 =0%), and the difference in TVR was also statistically significant in three RCT studies with follow-up periods of over 2 years (OR: 0.70, 95% CI 0.52 to 0.94; p=0.002, I 2 =15%), with values much lower in the BP-DES group (figures 5 and 6). ## Open access Safety endpoint: all-cause death, cardiac-related death, tVmi and St over follow-up periods of 1-5 years, 1 year and over 2 years All-cause death, cardiac-related death, TVMI and ST were considered the efficacy endpoints. The meta-analysis indicated no statistically significant difference between the two groups in all-cause death (OR: 0.88, 95% CI 0.72 to 1.07; p=0.20, I 2 =0%), cardiac-related death (OR: 0.89, 95% CI 0.71 to 1.12; p=0.32, I 2 =20%) and TVMI (OR: 0.73, 95% CI 0.53 to 1.01; p=0.05, I 2 =0%) over a follow-up period ranging from 1 to 5 years. Of the five studies that published 1-year data, all-cause death, cardiac-related death and TVMI were also not significantly different between the BP-DES and DP-DES groups ((all-cause death, OR: 0.91, 95% CI 0.71 to 1.15; p=0.42, I 2 =0%), (cardiac-related death, OR: 0.96, 95% CI 0.74 to 1.26; p=0.79, I 2 =35%) and (TVMI, OR: 0.73, 95% CI 0.53 to 1.01; p=0.05, I 2 =0%)). In the five studies with follow-up periods of over 2 years, similar findings were observed for the all-cause cardiac death, cardiac-related death and TVMI ((all-cause death, OR: 0.85, 95% CI 0.64 to 1.12; p=0.25, I 2 =0%), (cardiac-related death, OR: 0.77, 95% CI 0.56 to 1.17; p=0.12, I 2 =0%) and (TVMI, OR: 0.79, 95% CI 0.51 to 1.22; p=0.28, I 2 =0%)) (figures 7-9). However, the total ST incidence, including the definite ST, probable ST and definite or probable ST incidence, was significantly different between the BP-DES and DP-DES groups during the follow-up period (OR: 0.59, 95% CI 0.46 to 0.77; p=0.0001, I 2 =48%). Further analysis revealed no difference in total ST for the 1-year follow-up (OR: 0.61, 95% CI 0.32 to 1.15; p=0.13, I 2 =72%), while the meta-analysis indicated a statistically significant difference in the total ST for the follow-up Open access # Discussion The choice of stent in patients undergoing PCI for ACS is debated. Coronary intervention with second-generation DP-DES generally reduces the need for revascularisation and improves mortality compared with BMS and firstgeneration DP-DES. Furthermore, the risk of late ST with DP-DES tends to off-set these benefits, as seen in registries and clinical trials comparing DP-DES to BMS. BP-DES was designed to leave only the BMS behind once the polymer completely bio-degraded after drug elution and may represent an attractive solution for patients with ACS. [bib_ref] Drug-Eluting coronary stents: insights from preclinical and pathology studies, Torii [/bib_ref] Prior meta-analyses have compared the clinical outcomes among BMS, DP-DES and BP-DES in patients with stable coronary artery disease, but no previous metaanalysis of RCTs and prospective trials directly compared clinical outcomes between BP-DES and DP-DES for the treatment of ACS. To our knowledge, this meta-analysis exclusively compared BP-DES to DP-DES. It included 7 trials representing 8089 patients with relatively long follow-up durations, ranging from 1 year to 5 years. BP-DES have been hypothesised to offer improved outcomes, mainly in the long term; however, several prior meta-analyses have demonstrated different outcomes with Open access Target vessel myocardial infarction (MI). BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents. BP-DES compared with DP-DES in patients undergoing PCI. Bangalore et al observed that BP-DES were associated with higher mortality than DP-DES beyond 1 year of follow-up. [bib_ref] Bare metal stents, durable polymer drug eluting stents, and biodegradable polymer drug..., Bangalore [/bib_ref] El-Hayek et al demonstrated no significant difference in mortality between these stent types. [bib_ref] Meta-Analysis of randomized clinical trials comparing biodegradable polymer drug-eluting stent to second-generation..., El-Hayek [/bib_ref] In our study, there were no significant differences in MACEs, allcause death, cardiac-related death, TVMI, TVR or TLR at a follow-up period of 1 year and no significant differences in all-cause death, cardiac death or TVMI at a follow-up period of over 2 years. However, at a follow-up of over 2 years, MACEs, TVR and TLR were significantly lower in the BP group than those in the DP group. Pilgrim et al observed higher all-cause mortality among patients treated with BP-DES than with DP-DES in the BIOSCI-ENCE trial; they also observed comparable all-cause mortality rates among patients treated with BP-DES and DP-DES in the BIOSTEMI trial with a 2-year follow-up. [bib_ref] Meta-Analysis of randomized clinical trials comparing biodegradable polymer drug-eluting stent to second-generation..., El-Hayek [/bib_ref] Iannaccone et al observed that BP-DES might decrease the risk of ischaemic events in selected high-risk subgroups of patients, although the two DES stents share the same safety factors for patients in high-anatomical-risk settings like left main disease. [bib_ref] Comparison of bioresorbable vs durable polymer drug-eluting stents in unprotected left main..., Iannaccone [/bib_ref] Together, these findings suggest that BP-DES share similar outcomes in terms of MACEs (all-cause death, cardiac-related death, TVMI, TVR and TLR) during a 1-year follow-up and might show significantly improved clinical outcomes over a 2-year follow-up. ST is defined as a thrombotic occlusion of a coronary stentand is a major complication. The risk of ST, particularly late ST (occurring beyond 30 days), remains one of the major concerns limiting the use of DES in the treatment of ACS. [bib_ref] Early and late coronary stent thrombosis of sirolimus-eluting and paclitaxel-eluting stents in..., Daemen [/bib_ref] Early-generation DP-DES were associated with increased rates of very late (>1 year) ST compared with BMS. It was hypothesised that the mechanism underlying late ST with first DP-DES in ACS was related to adverse reactions with the DP, [bib_ref] Late incomplete apposition after drug-eluting stent implantation: incidence and potential for adverse..., Siqueira [/bib_ref] and the use of more biocompatible polymers has been associated with a reduction in ST in high-risk patients. [bib_ref] Everolimus-Eluting stent versus bare-metal stent in ST-segment elevation myocardial infarction (examination): 1..., Sabate [/bib_ref] In the LEADERS trial, the rate of very late ST was lower with the use of the BP-DES than that with DP-DES. [bib_ref] Biodegradable polymer drug-eluting stents reduce the risk of stent thrombosis at 4..., Stefanini [/bib_ref] Our data demonstrated that both BP-DES and DP-DES have similar risks of ST beyond 1 year. However, BP-DES are associated with a significantly reduced risk of ST at a follow-up of over 2 years compared with DP-DES (OR: 0.64, 95% CI 0.46 to 0.88; p=0.006, I 2 =0%). In contrast, Kim et al observed that the incidence of ST by groups demonstrated numerically lower rates in the DP-DES group (0.1%) than those in the BP-DES group and that all late ST cases occurred in those receiving thick-strut BP-DES stents. They proposed that no meaningful differences in terms of ST could be identified between the different polymer technologies by intravascular imaging and that the association of polymer technology and the risk of the ST was difficult to prove. [bib_ref] Durable polymer versus biodegradable polymer drug-eluting stents after percutaneous coronary intervention in..., Kim [/bib_ref] Therefore, it may be hypothesised that BP-DES result in improved arterial healing, which not only minimises the risk of ST, but also improves the long-term durability of the antirestenotic efficacy in the long term, although the two groups have a similar risk of ST beyond 1 year. # Limitations The present study had several limitations. First, this study included RCTs and shares the limitations of original studies. Second, BP-DES are a heterogeneous group of stents, differing in stent platform thickness, time to complete degradation of the polymer and drug-elution kinetics. DP-DES are equally heterogeneous groups. Innaccone et al observed that lower strut thickness would have a positive clinical outcome, thereby reducing ST and TLRs. [bib_ref] Impact of strut thickness and number of crown and connectors on clinical..., Iannaccone [/bib_ref] We were unable to match the stents with regards Open access to the strut thickness. Consequently, the reported results may not be generalisable to all stents from the respective group. Third, over 6 months of dual antiplatelet therapy (DAPT) was provided to the patients in our study, including those in RCTs. D'Ascenzo et al observed a similar rate of MACEs between durable and BPs, irrespective of DAPT length, and the DAPT duration seems to partially impact the risk of adverse events of different types of stents during follow-up. [bib_ref] Impact of design of coronary stents and length of dual antiplatelet therapies..., D&apos;ascenzo [/bib_ref] Thus, we remain concerned that the duration differences of DAPT may influence the clinical outcomes. # Conclusion In this meta-analysis comparing BP-DES to DP-DES in patients with ACS who underwent PCI, the data indicated that both polymer types demonstrated excellent safety and efficacy profiles at 1 year. There was a slightly increased incidence of MACEs, TLR, TVR and ST in the DP-DES group in the follow-up period of over 2 years, suggesting that BP-DES may be more favourable for treating patients with ACS. These findings should be confirmed by longterm follow-ups in RCT trials. # Data availability statement No additional data is available. ## Patient and public involvement We did not require patient and public involvement, as this is a meta-analysis, and no new patients were enrolled in the study. Contributors CH, ZW and HY developed the idea of the study, participated in its design and coordination and helped draft the manuscript. TL and TW contributed to the acquisition and interpretation of data. YZ and YL provided a critical review and substantially revised the manuscript. All authors read and approved the final manuscript. funding This work was supported by the Hunan Provincial Natural Science Foundation of China (grant number 2020JJ4787). Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. Patient consent for publication Not applicable. ethics approval Not applicable. Provenance and peer review Not commissioned; externally peer reviewed. data availability statement No data are available. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. open access This is an open access article distributed in accordance with the Creative Commons Attribution Non Commercial (CC BY-NC 4.0) license, which permits others to distribute, remix, adapt, build upon this work non-commercially, and license their derivative works on different terms, provided the original work is properly cited, appropriate credit is given, any changes made indicated, and the use is non-commercial. See: http://creativecommons.org/licenses/by-nc/4.0/. orCid id Can Huang http://orcid.org/0000-0002-8659-9178 referenCeS [fig] Figure 1: Baseline characteristics and stent information of patients with acute coronary syndrome (ACS). BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents. [/fig] [fig] Figure 2: Baseline characteristics and stent information of patients with acute coronary syndrome (ACS). BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents; LVEF, left ventricular ejection fraction. [/fig] [fig] Figure 3: Baseline characteristics and stent information of patients with acute coronary syndrome (ACS). BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents [/fig] [fig] Figure 4: Primary endpoint: major adverse cardiac events (MACEs). BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents. [/fig] [fig] Figure 5: Target vessel revascularisation (TVR). BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents. the removal of these articles, 92 clinical studies and RCTs of the 2 polymers remained. After reading the full texts, 28 articles about ACS remained, with 20 articles including chronic and ACS. Finally, 8 articles, with 7 RCTs, comparing BP-DES and DP-DES in patients with ACS were identified and included in the qualitative and quantitative analyses. 19-26 The follow-up duration ranged from 1 year to 5 years (online supplementary table 1 and online supplementary material 2). general features of the trials A total number of 8089 patients (3898 patients who were treated with BP-DES and 4191 patients who were treated with DP-DES) were included in this analysis. Further details about the quality of RCTs; total number of patients retrieved from each trial; publication years; countries of origin of the publications; centres in which the trials were performed; follow-up durations; risk factors; and primary, efficacy, and safety endpoints are listed in online supplementary table 2 and online supplementary material 2. [/fig] [fig] Figure 6: Patient characteristicsThe baseline features of the patients are summarised in online supplemental table 2. The mean age of the patients who were treated by BP-DES ranged from 61.3 to 64 years, whereas the mean age of the patients who were treated by DP-DES ranged from 61.7 to 64.1 years. The proportions of male patients were above 70% in all included trials. There was no difference in age (mean difference (MD): 0.14, 95% CI −0.66 to 0.38; p=0.60, I 2 =0%), sex (male) (OR: 1.10, 95% CI 0.99 to 1.23; p=0.07, I 2 =0%), hypertension (OR: 1.03, 95% CI 0.94 to 1.13; p=0.57, I 2 =37%), dyslipidaemia (OR: 0.92, 95% CI 0.83 to 1.02; p=0.10, I 2 =36%), LVEF (MD: 0.00, 95% CI 0.00 to 0.01; p=0.12, I 2 =12%), body mass index (MD: 0.07, 95% CI −0.11 to 0.25; p=0.44, I 2 =0%), diabetes (OR: 0.92, 95% CI 0.83 to 1.02; p=0.13, I 2 =21%), total stent length (MD: −0.72, 95% CI −2.30 to −0.85; p=0.37, I 2 =40%) and in the number of stents per person (MD: −0.00, 95% CI −0.05 to 0.04; p=0.84, I 2 =0%) among patients who were Open access Target lesion revascularisation (TLR). BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents. [/fig] [fig] Figure 7: All-cause death. BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents. [/fig] [fig] Figure 8: Cardiac-related death. BP-DES, biodegradable polymer drug-eluting stents; DP-DES, durable polymer drug-eluting stents.periods of over 2 years (OR: 0.63, 95% CI 0.47 to 0.85; p=0.002, I 2 =0%) (figure 10). [/fig]

No Difference in Small or Large Nerve Fiber Function Between Individuals With Normal Glucose Tolerance and Impaired Glucose Tolerance OBJECTIVEdTo assess small and large nerve fiber function in people with normal glucose tolerance (NGT), impaired glucose tolerance (IGT), and type 2 diabetes (T2D).RESEARCH DESIGN AND METHODSdParticipants were recruited consecutively from a population-based cohort: NGT (n = 39), IGT (n = 29), and T2D (n = 51). Electrophysiological measures included nerve conduction studies and thermal thresholds. Intraepidermal nerve fiber density (IENFD) in skin biopsies was calculated.RESULTSdThere was no difference between IGT and NGT in sural nerve conduction, IENFD, and thermal thresholds. IENFD was significantly lower in T2D (median = 2.8 fibers/mm [interquartile range 1.1-4.7 fibers/mm]) than NGT individuals (4.5 fibers/mm [3.4-6.1 fibers/mm]; P , 0.05). T2D participants had poorer nerve conduction and higher heat thresholds than NGT and IGT.CONCLUSIONSdLarge and small nerve function in people with IGT did not differ from those with NGT. Our finding does not support the existence of neuropathy in a prediabetic stage. A high prevalence of impaired glucose tolerance (IGT) in individuals with idiopathic neuropathy has been reported (1), but whether neuropathy already exists in the prediabetic stage, i.e., IGT, is unknown [bib_ref] Does impaired glucose metabolism cause polyneuropathy? Review of previous studies and design..., Dyck [/bib_ref]. In a population-based study, neuropathy was marginally more common in IGT than in normoglycemic controls [bib_ref] Prevalence of polyneuropathy in pre-diabetes and diabetes is associated with abdominal obesity..., Ziegler [/bib_ref] , but others reported no difference in measures of neuropathy between IGT and normal glucose tolerance (NGT) [bib_ref] Impaired glycemia and diabetic polyneuropathy: the OC IG Survey, Dyck [/bib_ref]. When addressing the question of whether "IGT neuropathy" truly exists, objective measures of nerve dysfunction are frequently crude and focused on large nerve fibers, and small nerve fiber dysfunction is often overlooked [bib_ref] Prevalence of polyneuropathy in pre-diabetes and diabetes is associated with abdominal obesity..., Ziegler [/bib_ref] [bib_ref] Impaired glycemia and diabetic polyneuropathy: the OC IG Survey, Dyck [/bib_ref]. Thus, our aim was to study measures of both small and large nerve function in well-characterized normoglycemic, IGT, and type 2 diabetic (T2D) individuals. ## Research design and methods ## Study population The study population, their glycemic status verification, and other possible causes of neuropathy were considered and have been described earlier [bib_ref] Heat shock protein 27 is associated with better nerve function and fewer..., Pourhamidi [/bib_ref]. All individuals gave informed consent to participation. The regional ethical review board of Umeå University approved the study. ## Measurements Blood samples were drawn and measured for cholesterols, triglycerides, creatinine, fasting plasma glucose, and HbA 1c . Anthropometry and other measurements have been described elsewhere [bib_ref] Heat shock protein 27 is associated with better nerve function and fewer..., Pourhamidi [/bib_ref]. Neurophysiological assessment Nerve conduction. Standardized motor and sensory nerve conduction studies were performed on the right peroneal and sural nerve by a neurophysiologist blinded to the individuals' group identity. Thermal threshold testing Thermal threshold tests were performed with Thermotest equipment (Somedic AB, Hörby, Sweden) by using the method of limits [bib_ref] Reproducibility and influence of test modality order on thermal perception and thermal..., Heldestad [/bib_ref]. ## Skin biopsy Thin skin biopsies (5 mm) were taken for microscopical assessment. Procedures were developed (9) and modified (10) from published guidelines [bib_ref] European Federation of Neurological Societies; Peripheral Nerve Society. European Federation of Neurological..., Lauria [/bib_ref]. The intraepidermal nerve fiber density (IENFD) denotes the number of fibers per millimeter of epidermal length (mean counts in three sections). Intra-and interobserver reliabilities were r s = 0.98 and 0.84, respectively. ## Statistical analyses Data are presented as numbers (n) and proportions (%), and distribution as mean and SD or median and interquartile range (IQR). Differences between groups were tested by ANOVA and subsequent Student t test for normally distributed variables. For nonnormally distributed variables, the Kruskal-Wallis test was applied with subsequent Mann-Whitney U testing. A P value , 0.05 was considered statistically significant. Statistical analyses were performed with SPSS 19 (SPSS Inc., Chicago, IL). # Results ## Baseline characteristics Clinical characteristics of the 119 participants are presented in [fig_ref] Table 1: but was significantly lower in T2D compared with NGT [/fig_ref]. Ages were similar in all three groups. People with IGT showed no significant metabolic differences compared with NGT, whereas patients with diabetes had metabolic perturbations compared with both NGT and IGT [fig_ref] Table 1: but was significantly lower in T2D compared with NGT [/fig_ref]. ## Nerve conduction Sural nerve conduction did not differ between IGT and NGT. No difference was seen in sural amplitude between the groups [fig_ref] Table 1: but was significantly lower in T2D compared with NGT [/fig_ref]. People with IGT had a lower conduction velocity (CV) of the peroneal nerve than those with NGT. The CV of the peroneal and sural nerve was lower in T2D patients compared with NGT individuals [fig_ref] Table 1: but was significantly lower in T2D compared with NGT [/fig_ref]. ## Thermal thresholds There were no differences in heat or cold thresholds between IGT and NGT [fig_ref] Table 1: but was significantly lower in T2D compared with NGT [/fig_ref]. The proportion of abnormal heat thresholds was significantly higher in individuals with T2D than NGT and IGT. ## Ienfd IENFD did not differ significantly between IGT and NGT ( CONCLUSIONSdIGT individuals did not show different large and small nerve fiber function compared with NGT. As expected, patients with T2D had poorer small and large nerve fiber function than NGT and IGT. ## Igt and nerve dysfunction It is not clear if neuropathy is found in the prediabetic individuals with IGT . A high prevalence of IGT in individuals with idiopathic neuropathy has been reported (1,12), but these were individuals with existing neuropathy and in whom glycemic status was subsequently assessed. The retrospective study design is less appropriate for ascribing IGT as a potential cause of neuropathy. A reduction in IENFD has been reported in individuals with diabetes without clinical or electrophysiological indications of nerve dysfunction [bib_ref] Intraepidermal nerve fiber density as a marker of early diabetic neuropathy, Umapathi [/bib_ref]. In addition, it has been reported that there is a loss of IENFD in individuals with IGT (14,15), suggestive of small nerve fiber dysfunction being present in a prediabetic stage. In a population-based study, neuropathy was marginally increased in IGT, but the measure of neuropathy was rather crude and mainly on large fibers (4). One recent similar study showed no difference between IGT and NGT (6); however, no detailed measures of small nerve fiber function, particularly IENFD, were assessed. # Limitations and strengths First, our study is limited by a relatively small group size, which probably reduced the power to detect differences in IENFD between groups. However, our study provides detailed assessment of nerve function in individuals with IGT and NGT without any trend in results suggesting differences between the two groups. Second, the cross-sectional design did not enable us to study cause and effect. Moreover, when assessing IENFD, we used thin sections of 5 mm as compared with thick 50-mm sections suggested by published guidelines. However, it still allows for group comparison between NGT, IGT, and T2D within our study, but hampers comparisons to studies using thicker sections. Our study has the following strengths: all individuals were recruited consecutively from a population-based sample, were well defined in terms of glycemic status with a strict definition of IGT based on two oral glucose tolerance tests, and were all of the same age. To avoid bias, neurophysiological measurements were performed by personnel blinded for the glucose status of participants. [table] Table 1: but was significantly lower in T2D compared with NGT. Women had higher IENFD than men (median = 4.8 fibers/mm [IQR 3.2-6.4 fibers/ mm] vs. 2.7 fibers/mm [1.6-4.7 fibers/ mm]; P , 0.001). However, there was no interaction between sex and small or large nerve fiber function (data not shown). [/table]

A novel phenotype of 13q12.3 microdeletion characterized by epilepsy in an Asian child: a case report Background: The microdeletion of chromosome 13 has been rarely reported. Here, we report a 14-year old Asian female with a de novo microdeletion on 13q12.3. Case presentation: The child suffered mainly from two types of epileptic seizures: partial onset seizures and myoclonic seizures, accompanied with intellectual disability, developmental delay and minor dysmorphic features. The electroencephalogram disclosed slow waves in bilateral temporal, together with generalized spike-and-slow waves, multiple-spike-and-slow waves and slow waves in bilateral occipitotemporal regions. The exome sequencing showed no pathogenic genetic variation in the patient's DNA sample. While the single nucleotide polymorphism (SNP) array analysis revealed a de novo microdeletion spanning 2.324 Mb, within the cytogenetic band 13q12.3. Conclusions: The epilepsy may be associated with the mutation of KATNAL1 gene or the deletion unmasking a recessive mutation on the other allele, and our findings could provide a phenotypic expansion. # Background Epilepsy refers to a chronic neurologic disorder, and it leads to impairments of cognitive and behavioral function. Although the mechanism of epilepsy is still unclear, it is believed that genetic cause is strongly associated with epilepsy of infancy and childhood. Moreover, the genetic cause of epilepsy was initially demonstrated in 2001, with a finding that all seven children in a study of Dravet syndrome had a de novo SCN1A mutation. With the development of molecular techniques, more discoveries between genetics and epilepsy have been revealed. Besides, chromosome 13 owns one of the lowest gene densities among human chromosomes and structural and functional variations of it may lead to 13q-syndrome, but interstitial deletion of 13q12.3 has only rarely been reported. M. Drummond-Borg et al.have reported a complex chromosome rearrangement involving chromosome 2,13, and 20 in the normal mother of a girl with mild clinical features, developmental delay and an interstitial deletion of 13q12.1-q14.1. Also, another 14 cases of de novo 13q partial deletions (seven terminal and seven interstitial) ranging from 4.2 to 75.7 Mb showed varying degrees of intellectual disability and specific clinical features, among them, 8 had central nervous system anomalies, 6 had eyes abnormalities, 9 had facial dysmorphisms and 10 had hand or feet anomalies. Der Kaloustian et al.have revealed a patient with an interstitial deletion of 2.1 Mb at 13q12.11 who had mild developmental delay, craniofacial dysmorphism, a pectus excavatum, narrow shoulders, malformed toes café-au-lait spots. Furthermore, a child with approximate 12 Mb deletion involving chromosome bands 13q12.3-13q14.11 showed immunodeficiency with elevated IgM levels, mild and transient cerebellar ataxia, and developmental delay. However, currently, there is no report illustrating the correction between microdeletion of 13q12.3 and epilepsy. Here, we present an Asian (Chinese) patient with a microdeletion of 2.324 Mb on 13q12.3, who displayed the recurrent unconsciousness with convulsion for 7 years. ## Case presentation The proband is a 14-year-old Asian female. She is the first child of nonconsanguineous parents who have another healthy child. The family history was unremarkable. She was delivered by forceps-assisted vaginal delivery at gestational age 40 weeks, with 20 h of labor. Her birth weight was 4250 g. She was not able to suckle for several days after she was born. A developmental delay was observed since her first few months of life. The child had the capability of independent walking at 17 months. At the age of 20 months, she was able to speak. The child occurred hyperpyretic convulsion twice at age of four and a half, and five respectively. At the age of 7 years, she initially appeared absence, unconsciousness, right skew of head and eyes, head back, tumble, limb convulsion, lips cyanosis, rustle in the throat, sustaining for 2 to 4 min, coupled with headache, emesis and impaired consciousness. This situation occurred 3 to 4 times per year. Electroencephalogram (EEG) was obtained at 8 years old, which showed frequent epileptic discharge in right medial temporal lobe at awake, significantly paroxysmal epileptic discharge in bilateral lobe during sleep, and sporadic epileptic discharge in central and superior lobe during sleep. Then the same attack occurred twice at the ages of 9 and 10 respectively. At the age of 11, the frequency of attack increased to four times per year, and even more regular at age of 12. Therefore, a second EEG was performed at her age of 12 and revealed epileptic discharge in occipital lobe, suspecting her of absence seizure. Meanwhile, brain magnetic resonance imaging (MRI) manifested signal enhancement in bilateral hippocampus on FLAIR sequence. Between 13 and 14 years of age inclusive, epileptic seizures took place about every 7 to 10 days, with rapid vibration of lower limbs occurring dozens of times per day. Thus, a third EEG was performed to the child that illustrated slow waves in bilateral temporal regions, significant in left, with generalized spike-and-slow waves, multiple-spike-and-slow waves and slow waves in bilateral occipitotemporal, significant in left. After admitting, the physical examination revealed specific facial features with ocular hypertelorism, low insertion of the columella, malar flattening, bend of index and ring fingers and transverse lines in both palms. Cognitive performance was unable to be assessed. Routine metabolic assays were performed the day after admission. Serum sodium valproate: 94.53μg/ml (reference value: 50.0-100.0μg/ml), thyrotropin: 8.30uIU/ml (0.34-5.6uIU/ml, mildly high), fibrinogen: 4.07 g/l (2.0-4.0 g/l, mildly high), alanine aminotransferase: 57iu/L (5.0-40.0iu/L, mildly high), indirect bilirubin: 2.99umol/ L (3.42-15.1umol/L, mildly low), γglutamyltranspeptidase: 57iu/L (7.0-50.0iu/L, mildly high), and blood ammonia: 70μg/dL (0.0-100.0μg/dL). Moreover, her abdominal ultrasound showed no obvious abnormality in her gall bladder, spleen, pancreas and kidney, while low echo was found in left lobe of liver, which indicated non-uniform fatty liver. The patient was diagnosed with two types of epileptic seizures: the first type of seizure was partial onset seizures characterized by recurrent absence, unconsciousness, convulsion of four extremities, and aconuresis. The partial onset seizures could last 2 to 4 min and occurred about every 7 to 10 days. The second type of seizure was myoclonic seizures, which manifested as rapid myoclonic jerks of bilateral lower limbs. The frequency of this type of seizure was dozens of times per day. She was provided levetiracetam (43.5 mg/kg/day), lamotrigine (3.0 mg/kg/ day), and sodium valproate (14.5 mg/kg/day). ## Genetic study # Methods Exome sequencing was performed. The whole area of exons and adjacent area of introns (50 bp) were captured by SeqCap EZ MedExome Kit (Roche Nimblegen) from segmented, spliced, amplified, and purified DNA sample obtained from peripheral blood of the patient. Then, the captured DNA was eluted, amplified and purified, later sequenced by Illumina. Moreover, comparison and identification of genetic variation was used Nextgene V2.3.4 software and UCSC hg19 human reference genomic sequence which collected the coverage of targeted region and average sequencing depth at the same time. The average sequencing depth of targeted area of whole exome sequencing was 100.05X, among which the sequencing depth was more than 20X in 95.35% targeted sequence. Besides, Sanger sequencing was applied to verify the genetic variation reported by abovementioned approach. SNP array was conducted by Infinium Global Screening Array. Hence, chromosome abnormalities such as the heteroploidy, deletion, repetition, and uniparental disomy of chromosome fragment, of autosomes and sex chromosomes were detected. # Results The exome sequencing was normal that no pathogenic genetic variation was detected in the patient's or her mother's DNA sample (Supplementary. The SNP array (details inrevealed a microdeletion with an approximate size of 2.324 Mb on the 13q12.3 region (arr [hg19] 13q12.3(29,376,209-31,700, 395) × 1), which included 9 unique genes: MTUS2, SLC7A1, UBL3, KATNAL1, HMGB1, USPL1, ALOX5AP, MEDAG3, and TEX26. However, the mutation was not found in her parents, the pedigree of the proband is presented in. # Discussion and conclusions Here, we report a 14-year-old girl being the carrier of a 2.324 Mb microdeletion on 13q12.3 with a novel phenotype: epilepsy. The deletion of chromosome 13q12.3 has been reported only rarely and we merely find three related studies (details shown on. The clinical phenotypes were complex with facial dysmorphism, hand or feet anomalies, intellectual disability, development delay, and other anomalies. And most of them had poor prognoses. Moreover, an identical mutation site with our case has been found in a studythat three probands mainly presented intellectual disability, postnatal microcephaly, and eczema/atopic dermatitis, however, none of them showed epileptic seizures. Nevertheless, none of the genes contained in the deleted region are associated with a known monogenic phenotype. MTUS2 regulates a protein involved in cardiac hypertrophy and neural differentiation, which has been confirmed in mouse and chicken models. SLC7A1 is related to the function of endothelium and the decrease of nitric oxide (NO) level, it also presents a genetic susceptibility to spontaneous hypertension. USPL1 represents a third type of SUMO protease, and its expression increases in breast tumor tissue. ALOX5AP has been linked to modulate the leukotriene biosynthesis pathway and increase the risk for myocardial infarction, stroke and restenosis. The functions of UBL3, MEDAG3, and TEX26 were not clear. However, currently no evidence has demonstrated the association between these genes and epilepsy (refer to. We speculate genes that are highly expressed in central nervous system (CNS) are dosage sensitive, which may lead to the occurrence of epilepsy. HMGB1 and KATNAL1 are highly expressed in CNS. HMGB1 is a possible dosage-sensitive gene encoding a ubiquitous nonhistone chromosomal protein expressed in the brain, which regulates inflammatory responses leading to neuronal excitability and seizures. However, it presents a gain-of-function rather than loss-of-function effect. Both animal and human studies have demonstrated that overexpression of HMGB1 induces epilepsy via regulating TLR4/NF-κB and p38MAPK signaling pathways. Moreover, KATNAL1 was initially identified with a circadian deficit, it has a similar domain structure as KATN A1 and the amino acid sequence is 80% identical. Studies has confirmed that loss-of-function effect of KATNA1 performs reduction of neuronal migration, axonal elongation and axonal branching. A recent study has found its association with the development of neuronal function and behavior. As well as multiple morphological abnormalities and defects in neuronal migration and morphology were detected in KATNAL1 mutant mice. Therefore, we assume that the mutation of KATNAL1 is one of the reasons why the proband presents epilepsy. On the other hand, it is rather usual to have an epilepsy in children with chromosomal abnormalities, a possibility that this manifestation might be in fact due to the deletion unmasking a recessive mutation on the other allele also cannot be excluded. We describe a novel phenotype of 13q12.3 microdeletion, characterized by spontaneously recurrent epileptic seizures, as absence, unconscientiousness, convulsion, rapid vibration of lower limbs with facial and volar dysmorphism, intellectual disability, and developmental delay. Our findings could be a phenotypic expansion (a set of observed phenotypic features extended beyond those previously reported in association with a particular locus) of 13q12.3 microdeletion. However, the underlying mechanism between 13q12.3 microdeletion and epilepsy remains unclear, which requires further in vitro and/or in vivo studies. ## Supplementary information Supplementary information accompanies this paper at https://doi.org/10. 1186/s12920-020-00801-1.: The results of exome sequencing of the proband and her mother. Ethics approval and consent to participate This study was approved by the human research ethic committees of Beijing Hospital of Traditional Chinese Medicine. Written informed consent was obtained from the parents/guardians. ## Additional file 1 supplementary ## Additional file 2 supplementary ## Consent for publication Written informed consent was obtained from the parents/guardians for the publication of this study. ## Competing interests The authors declare that they have no competing interests.

The use of granulocyte colony-stimulating factor to increase the intensity of treatment with doxorubicin in patients with advanced breast and ovarian cancer. [bib_ref] Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells, Souza [/bib_ref] [bib_ref] Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells, Souza [/bib_ref] [bib_ref] In vitro and in vivo analysis of the effects of recombinant human..., Bronchud [/bib_ref] [bib_ref] Adnramycin (NSC-123127) in the treatment of advanced breast cancer studies by the..., Hoogstraten [/bib_ref] [bib_ref] Recombinant human granulocyte colony-stimulating factor: effects on normal and leukemic myeloid cells, Souza [/bib_ref] [bib_ref] Adriamycin therapy for advanced ovanran carcinoma recurrent after chemotherapy, Hubbard [/bib_ref] [bib_ref] High-dose doxorubicin: an exploration of the doseresponse curve in human neoplasia, Wheeler [/bib_ref] [bib_ref] Dose: a critical factor in cancer chemotherapy, Frei [/bib_ref] [bib_ref] High-dose doxorubicin: an exploration of the doseresponse curve in human neoplasia, Wheeler [/bib_ref] [bib_ref] A phase I-II study of intensive dose adriamycin for advanced breast cancer, Jones [/bib_ref] [bib_ref] Phase 11 study of recombinant human granulocyte colony-stimulating factor in patients receiving..., Thatcher [/bib_ref] [bib_ref] High-dose doxorubicin: an exploration of the doseresponse curve in human neoplasia, Wheeler [/bib_ref] [bib_ref] A phase I-II study of intensive dose adriamycin for advanced breast cancer, Jones [/bib_ref] [bib_ref] Quality of lsfe in cancer trials, Brinkley [/bib_ref] # Patients and methods ## Patients Twenty-one patients were entered in this study. 19 with progressive histologically proven metastatic breast cancer resistant to endocrine therapy and two with ovarian carcinoma recurrent after chemotherapy. Their median age was 53 (range 30-67), 15 received G-CSF and doxorubicin (Adriamycin, Farmitalia) given every 2 weeks for a total of three cycles. at the following doses: 75mgm-2 (4 patients), OOmgm -2 (5 patients). 125 mgm -2 (6 patients). Two more patients received G-CSF and 150mgmof doxorubicin for a total of two cycles only. Four patients were treated with conventional doses of 75mg m -2, without G-CSF, as controls; it was planned that they should be treated every 2 weeks if the neutrophil count rose to more than 2.5 x 109 1 at this time. The treatment plan is shown in and pre-treatment patient characteristics are summarised in [fig_ref] Table I: RT, radiotherapy [/fig_ref]. None of the 15 patients with metastatic breast cancer treated with G-CSF had received previous chemotherapy, but all had received previous radiotherapy (to less than 50% of their active bone marrow). Eleven of these patients had more than two sites of disease and all had considerable tumour burden as assessed both clinically and radiologically [fig_ref] Table I: RT, radiotherapy [/fig_ref]. The two patients with recurrent ovarian carcinoma were previously treated with cisplatinum and cyclophosphamide containing regimens. All patients had measurable and/or evaluable disease and a performance score of 0-3 on the WHO scale. Exclusion criteria included any history of congestive heart failure or significant arrhythmias, previous anthracycline therapy, significant mediastinal irradiation, and a serum bilirubin >25 rmol 1 1, Clinical and laboratory monitoring Before each course of chemotherapy all palpable or superficial lesions were measured in two perpendicular diameters and visible lesions photographed. Base line studies included a chest radiograph, an isotopic bone scan with radiographs of areas of increased uptake, and haematological and biochemical screens. Isotopic liver scans were performed in patients with abnormal liver function tests. All patients underwent resting multiple-gated acquisition (MUGA) scans before each course of chemotherapy, after completion of therapy and 2-4 months later. The ejection fraction (LVEF) was calculated from the volume change in the left ventricle using standard methods and the pretreatment value was required to be >40% (minimal normal value at our centre). Electrocardiograms were performed before starting chemotherapy and when indicated. Patients were managed as outpatients attending a day ward clinic three times a week for blood counts for a total of 6 weeks. Doxorubicin was infused via a central vein catheter in 250ml saline over 30 min in order to minimise peak levels. Recombinant human G-CSF was supplied by AMGEN (Thousand Oaks, CA, USA) and was administered as a continuous infusion as previously described . The infusion pump (CADD-1 model, Pharmacia) was programmed to give lI0gkg-I day-1 of G-CSF from day 1 after chemotherapy to day 8 and 5ug kg 1day-I from day 8 to day 11 of each cycle. This was followed by 2 days without growth factor to allow normalisation of peripheral blood counts. Non-cardiac toxicities were documented according to WHO scores and, if visible, photographed. Patients were given prophylactic antiseptic mouthwashes and warned about possible mucositis. Patients were asked to complete the Rotterdam Symptom Checklist (de [bib_ref] Klachtenlijst voor Kankerpatienten. Eerste ervaringen, De Haes [/bib_ref] , a self-rating scale designed to measure psychological status (depression and anxiety), physical complaints (symptoms of disease and treatment toxicity) and functional status (ranging from personal care to going out shopping). Questionnaires were administered by a specialist nurse at study entry, on completion of chemotherapy and 2 months later. The psychological subscale of the questionnaire had been validated in women with advanced breast cancer, attending the same clinic, and an appropriate cut-off score established. I Scores > 10 suggest clinically important levels of depression and anxiety (Hopwood et al., in preparation). Functional status was quantified as per cent of the maximum disability score. Pain and shortness of breath were analysed individually, using a scoring of 0 (not at all), 1, 2 and 3 (severe). All patients were evaluated for response according to . Time to progression was from the beginning of chemotherapy until either new lesions appeared or any one existing lesion increased by 25% or more above its smallest size recorded. Disease recurrence in the complete responders was documented by biopsy. Serum levels of mucin-like carcinoma-associated antigen (MCA) were monitored in all patients by a two-step solid phase enzyme immunoassay with a monoclonal mouse antibody (MCA EIA Kit, Hoffman-La Roche & Co. Ltd, Basle, Switzerland). Pre-treatment serum levels above 11 U ml-' were regarded as positive. Similarly, CA 15-3 levels were also measured in the same serum samples by a solid phase two sites immunoradiometric assay (ELSA-CA15-3 kit, 1988, CIS Bioindustries, Gif-sur-Yvette, France) and pre-treatment serum levels above 20 U ml-I were regarded as positive. ## Pharmacokinetics of doxorubicin The pharmacokinetics of doxorubicin were determined in 11 patients (three patients at each dose level and two at 50mg m-2) by a high performance liquid chromatography (HPLC) assay following the first dose, essentially as described by [bib_ref] Liquid chromatographic analysis of adriamycin and metabolites in biological fluids, Israel [/bib_ref] and will be discussed more fully in a subsequent paper. # Statistical methods Response rates and toxicities were analysed in the 17 patients who had received G-CSF and doxorubicin. The nine patients who had received the lower doses of doxorubicin (75 and 00 mgm-2) were compared with the eight patients who had received the higher doses (125 and 150mgm-2). Becaue the numbers were small Fisher's exact test was used. Functional status scores were analysed by the Wilcoxon matched-pair signed rank test and anxiety-depression scores by McNemar's test, comparing the original pre-treatment scores with those recorded 2 months after completion of therapy. ## Resus Anti-tumour effects The results of treatment are shown in [fig_ref] Table I: RT, radiotherapy [/fig_ref]. At 75 and lOOmgm-2, 5/8 patients (62%) with metastic breast cancer achieved an objective regression, one being complete (12%). At 125 mgm-2 and 150 mgm-2 7/7 patients with metastatic breast cancer responded, four of them (57%) achieving complete remission. The patient with ovarian carcinoma present in each of the latter two groups also achieved a partial response, and so did 2/4 breast cancer patients in the control group (not treated with G-CSF). There were comparatively more complete responders in the two higher doses group (P=0.086), but this was a pilot study and the numbers were necessarily small. Nevertheless, an overall response rate of 80% (12/15) in patients with advanced breast cancer treated with G-CSF and a median time to progression of 6 months, range 2-9 months [fig_ref] Fgwe 2: Time to progression [/fig_ref] , are encouraging. Two patients, numbers 3 and 6, died from progressive disease within 6 weeks of therapy. Both had a very poor pre-treatment performance score (WHO grade 3) and failed to achieve significant regression of tumour burden. Similarly, one patient (number 19) in the control group, not given G-CSF, died after one course of chemotherapy from progressive disease. Tumour responses were 11 I)n _ 120' profile of the control group. Shaded areas represent the total area of neutropenia (ANC <1,000 Mm-3) following the first doxorubicin dose at 75mgm-2. b, Median ANC of patients receiving G-CSF and doxorubicin at 125 mgm-2 (0, six patients) and 150mgm-2 ([C1, two patients). -z also documented by measuring serum levels of tumour markers. Three of five patients who achieved a complete response had significantly raised pre-treatment levels of MCA, which in each case returned to normal within 2 months. A similar fall was also found in the CA15-3 levels of these three patients. Following disease progression/relapse patients were offered further therapy with cyclophosphamide, methotrexate and 5-fluorouracil. Seven patients are now assessable for response to second line chemotherapy and three have obtained objective responses (42%). Haematological changes Total and differential white counts were measured three times per week during the study. The absolute neutrophil counts (ANC) are shown in . The ANC rose to normal or above normal levels by day 12-14 at all dose levels of doxorubicin given with G-CSF infusions, whereas an ANC > 2.5 x 109 1O-1 was not reached until day 19-21 after 75mgm-2 of doxorubicin given without G-CSF. G-CSF infusion was followed by a rapid increase in peripheral neutrophil counts up to 40 x 1091 -1. The nadir was usually on day 7 post-chemotherapy when G-CSF was given, but it occurred on days 12-13 when G-CSF was not given. The nadir and duration of neutropenia were increased for the two higher dose levels of doxorubicin compared with the two lower ones . At 125 mgm-2 recovery from neutropenia following the third cycle of chemotherapy was slower than following the first two cycles. Platelet transfusions were required by 4/8 patients at the two higher dose levels, but no bleeding complications were seen. Blood transfusions were required by 4/9 patients in the two lower doses group and by 7/8 in the other, but in no case did the haemoglobin drop below 8 g dl-'. There were no lifethreatening infections, but 7/8 patients receiving 125 and 150mg m -2 required intravenous antibiotics at least once because of pyrexia and mucositis. Only one pyrexial episode was associated with positive blood cultures. ## Non-haematological toxicities Overall toxicities are represented in as the percentage of patients experiencing significant side-effects by the end of the full treatment period. There was a marked difference between the group of patients at the lower dose levels of doxorubicin and the rest . The former were managed as outpatients with the exception of overnight admissions for a blood transfusion at the completion of chemotherapy in four and one admission for an infective episode in two . In contrast, 7/8 a 100 75 cn CD 50 2-25 0 K n n patients given the two higher dose levels (125 and 150 mgm-2) had to be admitted. All patients developed mucositis resulting in three patients requiring parenteral nutrition, and in two cases this toxicity caused a 1 week delay in the final cycle of chemotherapy. The difference in the severity of mucositis between the two groups was statistically significant (P=0.002). New epithelial toxicities were also found. The most dramatic one was the development of erythema of palms and soles by 7/8 patients in the higher dose group, which in some cases progressed to superficial blistering and desquamation. Again, the difference between the two groups was statistically significant (P<0.005). A milder epithelial reaction also involved the vulva and perineum of 3/8 patients associated with vaginal candidiasis in two. Diarrhoea was reported by some patients but it was always tolerable and easily controlled. All these epithelial toxicities clared within 2 weeks. No patient-has developed clinical cardiotoxicity. The mean resting LVEF for all patients was 44% (range 40-60%) before therapy, 48% after receiving 225mgm-2, 45% after receiving 300mgm -2 and 39% at 375mgm-2. Two patients at the latter cumulative dose dropped their LVEF by 15%, but none have developed clinical signs of cardiac failure. ## Quality of life Fourteen patients receiving G-CSF completed quality of life questionnaires before, immediately after chemotherapy and, again, after a further 2 months. Eight scored above the threshold of the psychological subscale before treatment, and in five of these the scores increased at the end of chemotherapy, indicating worsening of depression and/or anxiety. However, all but one patient recorded scores within the normal range 2 months after the completion of treatment (P<0.05). Thirty-five per cent of patients had normal functional status before therapy. Of the remaining 65%, functional status improved significantly in 42%, did not change in 15% and deteriorated in 8% (P<0.05). Twelve of 14 patients reported pain before therapy and 10 of these patients had improved by 2 months after it. Similarly, 5/7 patients who had complained of breathlessness in the original questionnaire reported an improvement in the last one. Doxorubicin pharnacokinetics There was a linear increase in the area under the time-drug concentration curve (AUC) over the full dose range used. The kinetic parameters obtained suggest that there is no change in drug distribution with increased dose [bib_ref] In vitro and in vivo analysis of the effects of recombinant human..., Bronchud [/bib_ref]. Fugwe 4 Toxicities (WHO coded) represented as percentage of patients developing cytotoxic-related side-effects during and after chemotherapy. See text for explanation. a, Doxorubicin 75 or lOOmgm-2 every 2 weeks: 1, alopecia (WHO 2, 3); 2, nausea/ vomiting (WHO 2); 3, mucositis (V-1O 2); 4, mucositis (WHO 3); 5, mucositis (WHO 4); 6, diarrhoea (WHO 2); 7, i.v. antibiotics (WHO 2, 3); 8, platelet transfusions (no bleding); 9, red hands/feet (WHO 1); 10, vulvitis (WHO 2); 11, cardiotoxicity (WHO 1). b, Doxorubicin 125 or 150mgm-2 every 2 weeks: 1, alopecia (WHO 2, 3); 2, nausea/vomiting (WHO 2); 3, mucositis (WHO 2); 4, mucositis (WHO 3); 5, mucositis (WHO 4); 6, diarrhoea (WHO 2); 7, i.v. antibiotics (WHO 2, 3); 8, platelet transfusions (no bleeding); 9, red hands/feet (WHO 2); 10, red hands/feet (WHO 3); 11, vulvitis (WHO 2); 12, cardiotoxicity (WHO 1); 13, cardiotoxicity (WHO 2). Our previous study in patients with small cell lung cancer was the first to show that neutropemna and infection could be reduced by G-CSF following intermittent 'conventional' 3weekly chemotherapy . The study reported here shows that infusions of G-CSF allow dose escalations of chemotherapy and a decrease in the interval between doses. For example, if we take 300mgm-2 as the total dose administered, it would take a patient receiving 75 mgm-2 every 3 weeks a minimum of 9 weeks to complete therapy. This total dose can be administered in 4 weeks (lOOmgm-2 every 2 weeks) or in 2 weeks (lSOmgm-2) under G-CSF cover. This represents an increase in dose intensity of 2.2-4,5-fold. No more than 375 mg m-2 total dose could be given at this dose intensity because of doselimiting toxicities: severe mucositis and a severe desquamative skin rash in areas with.high epithelial turnover rate. In addition, some non-clinical cardiotoxicity was also seen at this cumulative dose. This distinct difference in toxicity between the two lower and the two higher doses of chemotherapy, particularly after the second and third cycles, made us consider whether the kinetics of doxorubicin were non-linear. However, examination of the pharmacokinetic profiles indicated a linear relationship between dose and AUC for the range of doses employed and intracellular accumulation of doxorubicin has been suggested as responsible for the increase in toxicities . None of the toxicities encountered were thought to be related to G-CSF therapy. An overall response rate of 80% in patients with advanced breast cancer, with a median time to progression of 6 months, was achieved in this preliminary phase II study. Response rates were probably higher at the higher doses, but the numbers were too small to reach statistical significance. Two months after doxorubicin-G-CSF therapy there was a pronounced improvement of symptoms compared with before treatment. These results are comparable, if not better, to those obtained with high dose chemotherapy and bone marrow transplantation [bib_ref] Advanced breast cancer highdose chemotherapy and bone marrow autotransplants, Antman [/bib_ref] [bib_ref] The role of high-dose chemotherapy with autologous bone marrow transplantation in the..., Hortobagyi [/bib_ref] , which is only available in a few specialised centres and carries a significant mortality (10-20%) and morbidity. In addition, bone marrow rescue is difficult to repeat and may not be appropriate in those with marrow involved with malignancy or in those without a suitable related donor. The importance of dose intensity has previously been reported for a variety of malignancies [bib_ref] Dose: a critical factor in cancer chemotherapy, Frei [/bib_ref] [bib_ref] A phase I-II study of intensive dose adriamycin for advanced breast cancer, Jones [/bib_ref] [bib_ref] Dose-response is alive and well, De Vita [/bib_ref] [bib_ref] The chemotherapy of lymphomas: looking back, moving forward. The Richard and Hinda..., De Vita [/bib_ref]. Many years ago Skipper showed that in experimental tumour models high dose intensity may make the difference between 50-100% cures and no cures at all, with equitoxic cytotoxic regimens [bib_ref] Criteria associated with destruction of leukemia and solid tumor cells in animals, Skipper [/bib_ref]. However, most of the clinical data on the impact of doseresponse in breast cancer come from retrospective studies, and there is a need for more clinical trials to assess the importance of both dose intensity and total dose in solid tumours . The availability of G-CSF will now allow direct testing of more intensive chemotherapy regimens in a variety of human cancers. The use of combination chemotherapy should further improve the clinical results of this new approach. It may give considerable benefits to patients with advanced cancer and possibly increase cure rates when used as an adjuvant to surgery. [fig] Fgwe 2: Time to progression (in months) for the 14 patients treated with G-CSF who achieved a partial or complete response to chemotherapy. Disease recurrence in the complete responders was documented by biopsy. a, Median absolute neutrophil counts mm -3 (ANC) of patients receiving 75mgm-2 of doxorubicin with (0) and without (A) G-CSF (four patients each) and lOOmgm-2 with G-CSF (El, five patients). The heavy line denotes the peripheral neutrophil [/fig] [table] Table I: RT, radiotherapy; CT, chemotherapy; NR, no response; PR, partial response; CR, complete response; B, breast primary; 0, ovarian primary. Patients 17-20 are controls, receiving no G-CSF. *CR in sites other than bone.Informed consent was obtained from all patients; the study protocol was approved by South Manchester ethical committee. [/table]

Thinopyrum ponticum Chromatin-Integrated Wheat Genome Shows Salt-Tolerance at Germination Stage A wild wheatgrass, Thinopyrum ponticum (2n = 10x = 70), which exhibits substantially higher levels of salt tolerance than cultivated wheat, was employed to transfer its salt tolerance to common wheat by means of wide hybridization. A highly salt-tolerant wheat line S148 (2n = 42) was obtained from the BC3F2 progenies between Triticum aestivum (2n = 42) and Th. ponticum. In the cross of S148 × salt-sensitive wheat variety Chinese Spring, the BC4F2 seeds at germination stage segregated into a ratio of 3 salt tolerant to 1 salt sensitive, indicating that the salt tolerance was conferred by a dominant gene block. Genomic in situ hybridization analysis revealed that S148 had a single pair of Th. ponticum-T. aestivum translocated chromosomes bearing the salt-tolerance. This is an initial step of molecular breeding for salt-tolerant wheat. # Introduction Salinity is a major external factor affecting plant growth and productivity. Of the world's 230 million ha of all irrigated lands, [bib_ref] Development of wheat-Lophopyrum elongatum recombinant lines for enhanced sodium "exclusion" during salinity..., Mullan [/bib_ref].5% are seriously affected by salinity (http://www.fao.org/ OPEN ACCESS nr/land/en/). Wheat is grown under irrigated and rain-fed conditions; both types of agriculture are threatened by salinization [bib_ref] Conventional and alien genetic diversity for salt tolerant wheats: Focus on current..., Mujeeb-Kazi [/bib_ref]. Therefore, the development of salt-tolerant wheat is an important breeding objective. To date, however, little progress has been made towards improving the salt tolerance of wheat. Shalaby et al. [bib_ref] Variation in salt tolerance among some wheat and triticale genotypes, Shalaby [/bib_ref] indicated that breeding salt tolerant wheat by exploiting naturally occurring variation is difficult. Fortunately, amongst halophytic members of Triticeae, tall wheatgrass species (e.g., Thinopyrum spp.) have received the most attention as a potential gene source for improving salt tolerance in wheat [bib_ref] Improving salt tolerance of wheat and barley: Future prospects, Colmer [/bib_ref] [bib_ref] Development of salinity-tolerant wheat recombinant lines from a wheat disomic addition line..., Wang [/bib_ref]. A perennial grass, Th. ponticum (Podp.) Barkworth and D.R.Dewey (2n = 10x = 70, JJJJsJs) was used as forage on salt-affected lands [bib_ref] Use of wild relatives to improve salt tolerance in wheat, Colmer [/bib_ref]. Several accessions of Th. ponticum survived 750 mM NaCl [bib_ref] High salt tolerance potential in wheatgrasses, Mcguire [/bib_ref] and some maintained reasonable growth at an EC of 13.9 dS·m -1 [bib_ref] Salt tolerance of twenty-five strains of Agropyron, Dewey [/bib_ref]. Thus, Th. ponticum is a promising material to transfer its salt tolerance into common wheat by chromosome engineering through wide hybridization. Towards the improvement of salt tolerance of wheat, wheat-tall wheatgrass amphiploids was developed as a new salt-tolerant cereal, named Tritipyrum (derived from Triticum spp. × Thinopyrum spp.) [bib_ref] Combined effect of salinity and hypoxia in wheat (Triticum aestivum L) and..., Akhtar [/bib_ref] [bib_ref] Tritipyrum, a potential new salt-tolerant cereal, King [/bib_ref] [bib_ref] Molecular cytogenetic characterization of parental genomes in the partial amphidiploid Triticum aestivum..., Brasileiro-Vidal [/bib_ref]. Although the resulting crop would not be expected to produce grain quality of bread or durum wheat, it could likely be used as feed wheat. The second step is to develop recombinant wheat lines containing small segments of tall wheatgrass chromosomes without linkage drag, such as deleterious effects on yield or grain quality. In this study, Th. ponticum chromosomes was transferred to T. aestivum by back crossing onto the amphiploids of T. aestivum × Th. ponticum, and that the salt-tolerance small chromatin was successfully inherited at germination stage in the BC4F2. # Results and discussion ## Transfer of th. ponticum genetic material with salt tolerance to wheat by wide hybridization Firstly, an amphiploid TAe68 (2n = 56) developed from the hybrid of T. aestivum salt-sensitive variety Jinmai 24 × Th. ponticum showed salinity tolerance. TAe68 was backcrossed to "Jinmai 24" twice. Until the BC2 crossing, the hybrid embryo was excised 15-20 days after pollination and cultured on the media for callus induction and then plantlets were differentiated. Through the BC2F2 to BC2F6 generation, plants grown in a greenhouse were selected according to the salt tolerance test. Chromosome numbers were examined using 2% (w/v) aceto-carmine, and Th. ponticum chromatins were identified by C-banding analysis according to Gill et al. [bib_ref] Standard karyotype and nomenclature system for description of chromosome bands and structural..., Gill [/bib_ref]. In the BC2F6 generation, a highly salt-tolerance with 2n = 42 was selected and backcrossed twice with T. aestivum salt-sensitive cv. Chinese Spring. The BC4F1 line showed 21 bivalents in pollen mother cells. These results indicated that BC3F2 line S148 is a Th. ponticum-T. aestivum translocation line. Grains of a salt-resistant line S148, Chinese Spring, and the BC4F2 progeny of Chinese Spring × S148 were used for genetic analysis for salt tolerance under NaCl conc. 0.1, 0.2, 0.3 and 0.4 M, respectively. Both genotypes germinated in the presence of 0.1 and 0.2 M NaCl, but subsequent coleoptile and root extension was retarded. Notably, only S148 was able to germinate in the presence of 0.4 M NaCl and showed the least reductions in coleoptile and root lengths [fig_ref] Figure 1: Salt tolerance test of Chinese Spring [/fig_ref]. In the cross of S148 × Chinese Spring, the BC4F2 seeds segregated into a ratio of 72 salt tolerant to 27 salt sensitive. In salt tolerant plants, the averaged coleoptile and root length were 17.5 and 21.4 mm, respectively. Whereas, in salt sensitive plants, averaged coleoptile and root length were 3.5 and 6.8 mm, respectively. This segregation ratio was well fitted to a ratio of 3:1 (χ 2 = 0.273; 0.50 < p < 0.75), indicating that the salt tolerance was conferred by a dominant gene block derived from Th. ponticum. ## Identification of th. ponticum chromatin in salt-tolerant recombinant wheat Fluorescence GISH method according to Tomita et al. [bib_ref] Revolver is a new class of transposon-like gene composing the Triticeae genome, Tomita [/bib_ref] was applied to detect the Th. ponticum chromatin in the wheat background. The total genomic DNA of Th. ponticum labeled with biotin-16-dUTP or Digoxigenin-11-dUTP was hybridized as the probe to the denatured chromosomes. In the double-exposed photographs, the in situ hybridization sites on Th. ponticum chromosome segments were visualized using FITC or rhodamine, and the other chromosomes were counter-stained with PI or DAPI [fig_ref] Figure 2: Genomic in situ hybridization [/fig_ref]. Using this approach, two alien chromosome segments were detected in S148 and some salt resistant individuals in BC4F2. GISH analysis verified that S148 had a single pair of Th. ponticum-T. aestivum translocated chromosomes including the salt tolerance gene block of Th. ponticum. The observed single genotypic ratio of salt tolerance means that this alien translocation transmitted normally and did not have segregation distortion. Soil salinity is a major abiotic constraint to agricultural productivity. Introgression of genes conferring salt tolerance from Th. ponticum (2n = 10x = 70) into Triticum aestivum is one of the few methods by which salt-tolerant wheat varieties can be developed. The modern breeding strategy is developing recombinant lines of wheat containing small segments of tall wheatgrass chromosomes without deleterious effects on yield or grain quality. Such an approach has enabled the use of tall wheatgrass to confer disease resistance to wheat [bib_ref] Wheat × Thinopyrum hybrids, Pienaar [/bib_ref] [bib_ref] Molecular aids for integration of alien chromatin through wide crosses, Fedak [/bib_ref] [bib_ref] Introgression of salt-tolerance from somatic hybrids between common wheat and Thinopyrum ponticum, Chen [/bib_ref] [bib_ref] Agropyron elongatum chromatin localization on the wheat chromosomes in an introgression line, Wang [/bib_ref] [bib_ref] Proteomic analysis on a high salt tolerance introgression strain of Triticum aestivum/Thinopyrum..., Wang [/bib_ref]. However, Th. ponticum-T. aestivum translocation lines with the salt tolerance have not been developed, and the salt tolerance of Th. ponticum has not been employed in wheat varieties. In this study, we attempted to identify alien chromatin in the progenies resulting from T. aestivum-Th. ponticum hybrids using genomic in situ hybridization method. Here, salt tolerance of Th. ponticum had been introgressed into bread wheat as a stably inherited translocated chromatin. This study is an initial step to developing the salt tolerant wheat plant. The future studies for line S148 will be further characterized by molecular cytogenetic approaches to reveal the specific chromosome arms of wheat and tallgrass which are involved to the translocation, as in the case of wheat-Lophopyrum elongatum recombinant lines [bib_ref] Development of wheat-Lophopyrum elongatum recombinant lines for enhanced sodium "exclusion" during salinity..., Mullan [/bib_ref]. Molecular genetic analysis of salt tolerance is also important [bib_ref] Proteomic analysis on a high salt tolerance introgression strain of Triticum aestivum/Thinopyrum..., Wang [/bib_ref] [bib_ref] Comparative transcriptome analysis of salt-tolerant wheat germplasm lines using wheat genome arrays, Mott [/bib_ref]. Salt tolerance in decaploid tall wheatgrass was associated with a capacity to restrict the rate of accumulation of sodium ion. Obtained Th. ponticum-T. aestivum translocation line with salt stress tolerance could potentially lead to identification of salt tolerance determinants. ## Experimental section ## Embryo culture The culture media for callus induction and callus maintenance are: (1) Murashige and Skoog (MS) medium + 200 mg/L glutamine + 100 mg/L asparagine + 600 mg/L hydrolytic lacto-albumin + 2 mg/L 2,4-D + 0.1 mg/L KT; (2) MS + 1 mg/L 2,4-D + 1 mg/L NAA + 0.1 mg/L KT; and (3) MS + 2 mg/L 2,4-D. The culture medium for differentiation is MS + 3 mg/L BA. Sugar 3%, agar 0.6%, pH = 5.6. Culture was maintained under a photoperiod of 14 h/day and temperature of 25 ± 1 °C. ## Salinity test The seeds were sown in Petri dishes on filter paper soaked with distilled water and 0.1, 0.2, 0.3 and 0.4 M NaCl and the dishes were placed in an incubator at 25 °C. After 7 days, coleoptile and main root lengths were measured. Electroconductivity was measured daily and the salinity was adjusted as needed to maintain the NaCl. ## Fluorescence genomic in situ hybridization The total genomic DNA of Th. ponticum was labeled with biotin-16-dUTP or Digoxigenin-11-dUTP (Roche Diagnostics, Basel, Switzerland), and was hybridized as the probe to the denatured chromosomes. After hybridization, biotin-labeled probe was detected by avidin-fluorescein isothiocyanate (FITC) with signal amplification using a biotinylated anti-avidin secondary antibody and fluorescein-avidin DCS (a cell sorter grade of fluorescein avidin D) (Roche Diagnostics). The Digoxigenen-labeled probe was detected by anti-Dig-rhodamine (Roche Diagnostics). Chromosomes were excited using a WBV or WIG filters (Olympus, Tokyo, Japan) and counter-staining with propidium iodide (PI) or 4',6-Diamidine-2'phenylindole dihydrochloride (DAPI) (Roche Diagnostics). [fig] Figure 1: Salt tolerance test of Chinese Spring (above) and S148 (below) at the germination stage. A, B, and C: NaCl conc. 0.0, 0.2 and 0.4 M, respectively. [/fig] [fig] Figure 2: Genomic in situ hybridization (GISH) results of line S148. (a) Two translocated chromosome segments (arrows) originated from Th. ponticum were visualized in a mitotic metaphase cell; (b) Two fluorochrome signals of Th. ponticum were detected in a mitotic interphase cell. Bars represent 10 μm. [/fig]

Realizing Innate Potential: CAR-NK Cell Therapies for Acute Myeloid Leukemia # Introduction Acute myeloid leukemia (AML), the most common acute leukemia in adults, is a biologically heterogenous disease. Genetic alterations driving proliferation or disrupting differentiation lead to the accumulation of immature blast cells, resulting in impaired bone marrow function. Standard anthracycline and cytarabine based chemotherapy is curative for a minority of patients, generally with low-risk disease identified by genetic, clinical and treatment response factors. Sensitive measurable residual disease (MRD) techniques often detect blast cells after treatment despite morphological remission and are ## Target antigens in aml Despite the absence of an ideal target, pre-clinical and several clinical reports exist of AML CAR-T therapies. CD33 is a well-established target, expressed in almost all AML cases, but exemplifies the challenges discussed above. Antigen negative sub-populations are ubiquitous and expression on myeloid precursors (and possibly HSCs) is challenging. Myelosuppression is encountered with the CD33 antibody drug conjugates (ADC) gemtuzumab ozogomycin and vadastuximab talirine and also with CD33 CAR-T cells. Transient partial response was documented in a clinical case report, although indications of intact hematopoiesis may reflect a low CAR binding affinity. CD123 is also widely expressed in AML and considered a more consistent LSC marker than CD33. Differing CAR designs interacting with low CD123 expression on HSCs may explain the varying myeloablative potential of CD123 CAR-T cells reported. Budde et al. described promising findings from 6 patients treated with CD123 CAR-T, including one complete response (CR). Although all participants were required to have an ASCT donor, myelosuppression without myeloablation was documented. Notably, half of the enrolled patients did not receive the planned treatment due to fatal infection, disease progression or failures in leukapheresis and manufacturing. C-type lectin-like molecule-1 (CLL-1, CLEC12A) has similar expression characteristics to CD123, but with less concern for myeloablation reflecting its absence on HSCs. CLL-1 CAR-T has been proposed as a consolidation strategy for AML in remission, as CLL-1 positive blast cells are relatively chemotherapy resistant and this approach reduced relapse in a xenograft model. The difucosylated carbohydrate antigen Lewis-Y was the target of the first clinical AML CAR-T trial conducted, where persistence of CAR-T cells without definite responses was documented and attributed to the low surface density of the antigen. Aberrantly expressed in AML, CD7 CAR-T is also in development, trading myelotoxicity for T-cell depletion and relevant for 30% of AML cases. Other approaches aim to adapt CAR-T to the antigenic landscape of AML. Targeting multiple antigens simultaneously may be fundamental to avoiding relapses from preexisting antigen negative sub-clones. Perna et al. compared proteomic and transcriptomic data from AML and normal tissues identifying antigen pairs to increase blast targeting without increasing toxicity to normal tissues. Additionally, immunophenotyping of a large cohort of AML cases identified antigen combinations of T-cell immunoglobulin and mucin-domain containing-3 (TIM-3) with either CLL-1 or CD33 as warranting further study. CAR technologies which restrict activation to cells expressing both antigens would restrict off tumor targeting to monocytes yet identify up to 75% of LSCs. A split, universal and programmable (SUPRA) CAR system with vast flexibility, enhanced precision and controllable dosing has been described in which the CAR scFv is replaced by an adaptor protein which then interacts with separate scFv containing constructs. The ability of this system to sequentially target multiple antigens without re-engineering T-cells cells ex vivo, and readily apply complex logic-based approaches to antigen combinations could be especially useful in the setting of AML. Indeed, the pre-clinical development and potential of a modular synthetic agonistic receptor T-cell platform using a similar principle and targeting CD33 and CD123 in AML has recently been described. This versatility will likely come at the cost of a requirement for repeated infusions reflecting the shorter half-life of a separately delivered scFv construct. While the prospectively identified combinations and novel split CAR approaches described above remain to be tested clinically there have been dual targeting approaches reported. Updated trial results of a dual CD33/CLL-1 CAR-T product which recognizes cells positive for either antigen were recently presented. CAR-T expansion and complete MRD negative remissions were achieved in 7/9 treated patients, with expected marrow suppression, permitting six patients to proceed to reduced intensity conditioning ASCT. An alternative approach to targeting multiple antigens uses the NKG2D receptor as a CAR binding domain. This activating receptor, present on T-cells and NK cells, recognizes 8 NKG2D ligands commonly expressed on AML blasts but not normal cell populations. NKG2D CAR-T cells have now entered clinical trials. Short term responses and poor persistence occur without LD chemotherapy, although successful bridging to ASCT was reported. Ongoing trials have been modified to include LD chemotherapy and a modified manufacturing technique supporting an early memory T-cell phenotype. A further alternative to eliminating off-target effects is to target neoantigen peptides presented at the cell surface in an HLA dependent fashion. This is the principle behind a promising HLA-A2 dependent, mutated nucleophosmin 1 (NPM1c) targeted CAR, applicable to 35% of AML cases. Others have sought to reduce expected off-tumor effects associated with target antigens. CRISPR/Cas9 editing has been deployed to knockout CD33 in HSCs, allowing CD33 negative (and CAR-T resistant) hematopoiesis post SCT in animal models. Transient CAR expression through mRNA delivery was evaluated in a phase I clinical trial reflecting concern for persistent off target effects using a CD123 CAR but also appeared to limit efficacy. Finally, modulation of CAR binding affinity allows for tumor specific targeting of antigens with overlapping but increased expression relative to normal tissues. This principle has recently been applied to CD38, an established target in multiple myeloma but also relevant for a proportion of AML cases. Despite these innovative and varied approaches, CAR-T therapies for AML remain investigational. For a detailed review of CAR-T in AML readers are referred to reference. ## Natural killer cells and aml ## Nk cell functions Natural killer cells are innate lymphocytes which express a collection of germline encoded activating and inhibitory receptors. The balance of signaling through these receptors determines the response of the cell, varying from tolerance (desired for most selfcell interactions), to triggering of a degranulation (perforin and granzyme) response. Target cell death can also be triggered through death receptors via NK cell expression of TNF-related apoptosis-inducing ligand (TRAIL) and Fas ligand. NK cells modulate broader immune responses through release of TNF-α and IFN-γ, the latter of which provides an important stimulus of dendritic cell maturation and thus adaptive immunity. NK cell TRAIL expression also contributes to immunomodulation through death receptor mediated elimination of specific T-cell and dendritic cell subsets. Important inhibitory signals to NK cells are delivered by inhibitory killer immunoglobulin-like receptors (KIR) and NKG2A, interacting with their ligands: epitopes present on specific subsets of MHC class I molecules. This system provides for self-tolerance and loss of HLA class I expression on virally infected and transformed cells is the basis of 'missing self' NK cell recognition. Although inhibiting mature NK cell responses, intact signaling through inhibitory KIRs and NKG2A during maturation is integral to the process by which NK cells ultimately acquire and calibrate their functional capacity, termed NK cell "licensing" or "education". Individuals frequently possess a population of hypofunctional NK cells expressing an inhibitory KIR for which the respective ligand is not present in their complement of self HLA molecules. Recruitment of this hypofunctional population by a strong activating stimulus has been recognized. In practice, loss of inhibitory stimuli alone is generally insufficient to trigger NK cell cytolytic activity. Activating receptors, such as natural cytotoxicity receptors (NCRs) NKp30, NKp44, NKp46 and the NKG2D receptor, contribute to NK cell activation in response to ligands induced with cellular stress on infected, damaged, or transformed cells. The strong activation signal provided by the FCγRIII (CD16) receptor binding to Fc portions of IgG antibodies on target cells overcomes intact inhibitory signals. This NK cell mediated antibody dependent cellular cytotoxicity (ADCC) contributes an important component of the clinical responses observed to anti-tumor monoclonal antibody therapies. NK cells are phenotypically and functionally heterogenous in vivo. A subset of therapeutic interest are adaptive NK cells which exhibit memory-like properties-enhanced responsiveness after an initial time-limited stimulus-a property which can be induced by combination cytokine exposure ex vivo to create 'cytokine induced memory-like' (CIML) NK cells. The unique characteristics of NK cells underly a role in a variety of homeostatic and disease processes including cancer immune surveillance and their growing application to cancer immunotherapy. ## Nk cells in aml immunoediting NK cells are activated by contact to AML blasts in vitro to varying degrees. If NK cell recognition and lysis toward AML in vivo were complete, the disease would not develop. It is evident that intact NK cell activity during disease development not only selects for AML blasts relatively resistant to NK cell targeting, but also processes capable of suppressing NK cell function. As a result NK cells isolated from patients with AML are functionally deficient relative to healthy persons and this is partially rectified for patients achieving complete remission with standard therapy. The quality of subsequent NK cell immune surveillance, reflecting both restoration of NK cell function and relative blast cell expression of ligands for activating and inhibitory NK cell receptors, is implicated in remission maintenance. Varied mechanisms underlying escape from NK cell activity in AML have been described, are summarized inand have recently been reviewed in detail. Adoptively transferred NK and CAR-NK cells are expected to rapidly encounter and potentially be inhibited by these same processes. The balance of signaling via traditional NK cell activating and inhibitory pathways is skewed in AML. MHC class I expression is generally maintained providing a baseline inhibitory stimulus to NK cells. NKG2D ligands are frequently down regulated including on LSC populations by a variety of mechanisms including proteolytic cleavage producing soluble NKG2D ligands. Upregulation of the immunosuppressive cell surface glycoprotein CD200 on AML blasts including LSCs is associated with reduced NK cell function through its receptor CD200R. The phenotype of NK cells is also modified. Expression of NCRs is reduced relative to healthy controls in an effect reliant on NK and blast cell contact. NKG2D downregulation occurs secondary to chronic exposure to hypoxia, cell surface and soluble NKG2D ligands and via blast and stroma derived TGF-β. NKG2A expression is often increased, at least partially driven by blast cell production of IL-10, while IFN-γ induces expression of the ligand for NKG2A (HLA-E), on blast cells. The DNAM1/TIGIT/CD96 axis is also impacted in AML. DNAM1 and TIGIT represent activating and inhibitory NK cell receptors, respectively for the antigen CD155 which can be expressed on AML blasts and MDSCs. The relative expression of the ligands and receptors of this family vary, but alterations have been implicated in suppressing NK cell responsiveness. Recently, sialic acid-based ligands for the NK cell inhibitory receptor siglec-7 have been recognized on CD43 (a sialoglycoprotein frequently expressed on AML blasts). Additional processes have been identified which suppress NK cell function in AML. Glycogen synthase kinase-3β (GSK3β) is upregulated in NK cells relative to healthy controls, and inhibits function through reduced expression of LFA-1 and impaired immune conjugate formation. Receptor activator of nuclear factor kappa-B ligand (RANKL, a TNF family protein) expression on blasts confers bidirectional immunosuppressive effects. Reverse signaling into blast cells leads to secretion of immunosuppressive cytokines including IL-6, while forward signaling via receptor activator of nuclear factor kappa-B (RANK) on NK cells directly inhibits NK responses. Persistent elevations in IL-6 during induction chemotherapy negatively impact prognosis in AML, which may reflect reduced expression of NK cell activating receptors and stabilization of malignant cell PD-L1 expression previously attributed to this cytokine. Blast cell production of soluble agonists activates the aryl hydrocarbon receptor (AHR) transcription factor in NK cells, driving microRNA29b expression, interfering with NK maturation and function. Reactive oxygen species (ROS) formation, driven by monocytic and myelomonocytic AML subtype expression of the NADPH oxidase component gp91 phox causes apoptosis of surrounding immune cells. Immune suppressive cell subsets are recruited and restrict NK cell activity. Myeloid derived suppressor cell (MDSC) expansion is facilitated by soluble NKG2D ligands, while regulatory T cell (Treg) expansion is supported by blast cell production of indoleamine 2,3-dioxygenase (IDO). These subsets inhibit NK cell activity through varied mechanisms including expression of membrane bound transforming growth factor beta (TGFβ) and limiting IL-2 bioavailability. MDSC populations upregulate CD155 in response to ROS, impairing traditional NK cell but not adaptive NK cell function reflecting differing TIGIT expression. Finally, hypoxia has been associated with varied inhibitory NK cell effects including reduced activating receptor expression and impaired production of IFN-γ. The transcription factor HIF1α is a clinically relevant mediator of hypoxia related NK cell inhibition in cancer. Thus, endogenous NK cells and those introduced by adoptive transfer face a complex network of inhibitory factors which need to be overcome to mediate anti-leukemic efficacy in AML. The relative importance of these mechanisms may vary between patients, between AML sub-clones, and from diagnosis to remission and relapse. ## Nk cells in asct The decision to pursue ASCT as consolidation therapy in AML considers relapse risk predicted from clinical, genetic and response related factors, versus procedural risks impacted by patient fitness and the degree of HLA matching of available donors. A multifaceted, immune mediated GVL effect confers partial protection against disease relapse after ASCT. T-cell reactivity occurs against minor HLA antigens or mismatched major HLA antigens, if present. This reactivity has traditionally been considered the major component of the GVL response and is closely allied with a risk of GVHD. NK cells also contribute to GVL responses in AML in a manner that is independent of GVHD, and to a greater extent than in other diseases treated with ASCT. NK cell GVL effects may reflect their 'natural cytotoxicity' or alloreactive recognition. The quantity of NK cells in HLA matched stem cell grafts has been correlated with relapse risk in AML, at least partially mediated by mature NK cells with high DNAM-1 expression with a potential to directly lyse CD112 and CD155 positive blast cells irrespective of alloreactivity. Alloreactive recognition of AML blasts by NK cells is most prominent after haploidentical stem cell transplant. In this setting, donor-recipient iKIR-KIR ligand mismatch occurs frequently, NK cells rapidly recover post-transplant and T-cell depletion of stem cell grafts is often performed to reduce GVHD risk. Thus, it is frequent to have 'licensed' donor derived NK cells expressing an iKIR for which the respective KIR ligand is missing in the recipient, supporting NK cell activation through 'missing self' reactivity. The presence of alloreactive NK cells in haploidentical grafts has been shown to robustly correlate with reduced disease relapse risk. However, haploidentical donors are not favored in general due to increased rates of GVHD and graft failure. For HLA matched donors, where iKIR-KIR ligand mismatch is less likely to occur, the presence of activating KIR2DS1 in donors has been implicated in a clinically relevant graft versus leukemia effect, modulated by the HLA-C phenotype of the donor. The presence of KIR2DS1 (which recognizes the HLA-C2 KIR ligand) predicts a reduced rate of relapse in matched, or one locus mismatch ASCT, if the donor is not homozygous for HLA-C2. This correlates with in vitro observations of tolerance induced by chronic HLA-C2 exposure, which is not observed with C1/C1 and C1/C2 haplotypes. Recruitment of the population of hypofunctional 'uneducated' NK cells expressing iKIRs for ligands found in neither donor nor recipient has also been proposed in the inflammatory milieu post ASCT and was also shown to impact the efficacy of histamine dichloride with IL-2 used as consolidation therapy. These observations have established the clinical relevance of NK cell alloreactivity in AML and provide an evidence base for donor choice in NK ACT and CAR-NK settings. ## Nk cell adoptive cell transfer for aml Rare but notable successes with early attempts at ACT immunotherapy, and growing recognition of the alloreactive potential of NK cells in ASCT led to the investigation of purified haploidentical NK cell ACT in AML. The development of LD chemotherapy regimens and exogenous cytokine support have been viewed as important components of this therapy. These elements were combined in the seminal description of haploidentical, short term activated NK ACT with systemic IL-2 support by . Subsequently a variety of approaches have been reported using short term activated or longer term expanded NK cells, from sources including apheresis product, HSC differentiated NK and irradiated NK cell lines. NK ACT has been investigated in two main scenarios outside the setting of ASCT across a range of single arm early phase clinical trials: in relapsed or refractory AML or as consolidation therapy for patients in remission. These accumulated reports of NK ACT offer insight into the factors associated with clinical responses and define the baseline activity on which CAR modified NK cell approaches seek to build. In their initial description, Miller et al. reported complete responses (CR) in 5/19 patients with relapsed/refractory AML, associated iKIR-KIR ligand mismatch with positive outcomes, and documented NK cell expansion in vivo (supported by intense LD chemotherapy and an associated rise in IL-15). Emphasizing the importance of the tumor microenvironment (TME) and reflecting concerns that systemic IL-2 can expand Treg populations, the group went on to demonstrate improved persistence and response rates of >50% with prior depletion of Treg cells using a recombinant IL-2-diptheria toxin protein. These impressive responses likely occurred rapidly as the earliest measures of chimerism and Treg depletion were correlative, and the window of IL-15 increase was brief. Improving NK cell persistence was identified as a priority. As an alternative approach to avoiding Treg stimulation the group utilized exogenous rhIL-15 in place of IL-2, demonstrating a 32-40% CR/CRi rate. In this study the relationship between responses and NK cell expansion in vivo was not observed, and CRS/ICANS was noted with subcutaneous, but not intravenous IL-15. Exogenous IL-15 however stimulates CD8+ T-cells introducing a concern for rejection of donor NK cells. Applying a similar LD and IL-2 supported approach in a group of AML patients in morphologic remission not considered eligible for ASCT, Curti et al. demonstrated an association between the percentage of alloreactive NK cells infused and subsequent relapse rates. Bjorklund et al. applied a lower intensity LD regimen and administered haploidentical NK cells without IL-2 support successfully documenting responses in de novo AML and MDS/AML. Rubnitz et al. reported application of a similar approach using systemic IL-2, and a lower intensity LD regimen in a pediatric setting, for intermediate risk AML in remission. The treatment was well tolerated and NK cell expansion occurred, although no benefit in preventing relapse was demonstrated in a small phase II trial. CIML NK cells administered after LD chemotherapy and with systemic IL-2 support robustly expand in vivo and also induce remissions in a proportion of patients with relapsed/refractory AML. Pre-clinical in vitro and xenograft models show enhanced anti-leukemic activity of CIML NK versus control NK cells. Irradiated NK-92 cells appear safe when applied as an off the shelf product, although lacked persistence and were of limited efficacy in a phase I trial. As an alternative cell source, Dolstra et al. described the use of NK cells differentiated from cord blood derived CD34 progenitor cells, as a remission maintenance strategy. The approach appears safe but low level chimerism of short duration was observed in the absence of cytokine support. These reports, and others, provide an insight into the factors which influence clinical responses to NK ACT in AML. Haploidentical NK cell ACT achieves complete responses for a subset of patients. Responses are generally short in duration but have provided bridging to potentially curative ASCT in some cases. In contrast to ASCT where alloreactive NK cell populations are replenished from donor derived hematopoiesis, available measures suggest haploidentical NK ACT leads to a limited window of expansion and persistence despite exogenous cytokine support. Notably, LD chemotherapy of varying intensity has been relied upon to support NK cell expansion, one parameter that has been associated with clinical responses. While immune mediated adverse effects have been few, those relating to marrow suppression post LD chemotherapy have been encountered. In certain cases, iKIR-KIR ligand mismatch is associated with responses and modulation of TME elements also appears to influence success. Evidence for immunoediting of residual blast populations has been documented. ## Car-nk: a compelling platform for aml immunotherapy? CAR-NK cells combine the antigen specific targeting of CAR-T cells with the innate activity of NK cell ACT creating a platform which overcomes many of the limitations of each of these therapies applied to AML. In the absence of an ideal CAR target antigen, the well-defined innate and alloreactive potential of NK cells against AML provides for activity against antigen negative subclones. NK cell ADCC in the presence of monoclonal antibodies could provide a simple and time limited dual targeting solution, with mechanistic similarities to the modular CAR designs in development. Several approaches to engineering NK cell persistence have emerged coupled with CAR-NK cell development, which could overcome a consistent limitation of NK ACT. These factors, along with a potential for 'off-the-shelf' application, and a safer adverse effect profile define an attractive approach to AML immunotherapy. CAR-NK cell application is also adaptable to both clinical scenarios discussed previously, with overlapping but distinct challenges. In relapsed/refractory disease, activity against bulk AML blasts is required, and maximal TME effects can be anticipated. Alternative therapies, where existing, are also generally myelosuppressive, and bridging to ASCT in this setting is often the established goalsupporting the use of myeloablative antigens. Rapid availability of CAR-NK products would be a distinct benefit here. For AML in remission, CAR-NK would be applied as a consolidation therapy initially for patients not considered candidates for ASCT. In this setting, there is a greater emphasis on LSC targeting, a diminished TME, but also a lower tolerance for persistent myelosuppressive effects beyond LD, as the alternative therapy may be observation or well-defined chemotherapy-based consolidation. Rapid availability is less essential, but tailoring therapy to a detected LSC immunophenotype may be feasible and could be combined with MRD based techniques to further select patients based on relapse risk. The versatility of CAR-NK therapies could provide solutions for each of these clinical scenarios. In this section we will consider the status of CAR-NK cell development for AML and remaining barriers to be overcome. ## Principles of car-nk therapies CAR-NK continues to represent a small segment of a landscape of CAR therapies dominated by T-cells. Active and completed clinical trials investigating CAR-NK cell therapies are summarized in, and have also been compiled in detail in recent articles. The pioneering phase I clinical trial at MD Anderson Cancer Center of cord blood derived CD19 CAR-NK cells represents the most advanced clinical data available and provides important insights for AML CAR-NK development. Using HLA-mismatched NK cells transduced with a transgene including a CD19 CAR, IL-15 and inducible caspase 9, Liu et al. demonstrated responses (including CR in 7/11 patients) in chronic lymphocytic leukemia and B-cell lymphomas without GVHD, CRS or ICANS. Notably, CAR-NK cells expanded and persisted for at least one year post infusion, perhaps reflecting autocrine IL-15 stimulation. Despite this persistence, CD19 positive relapses occurred without repeat expansion of CAR-NK cells. The authors report that this platform is capable of scaling to produce 100 doses from a single cord unit, which if successfully combined with cryopreservation would create a truly off-the-shelf product, and plan to expand the project across other diseases, including AML. Clinical reports of other CAR-NK therapy formats exist. Transient mRNA based NKG2D CAR expression was applied in primary NK cells for a clinical trial using local delivery to treat metastatic colorectal cancer with tumor responses reported. CAR NK-92 cell data has been reported in a small phase I trial (discussed below), and several other CAR NK-92 trials are ongoing. Human induced pluripotent stem cells (iPSCs) can be stably gene modified and subsequently differentiated into CAR-NK cells, creating a platform with several advantages. A master iPSC cell bank can be cryopreserved, harbor multiple gene edits and produce a homogenous final product. The iPSC platform is also compatible with non-viral transposon-based gene delivery systems, which to date have not been readily applied in primary NK cells. While current manufacturing protocols are longer than for other cell therapy products, the development of an ongoing manufacturing pipeline would allow for off-the-shelf application. Similarly to cord blood derived NK cells, iPSC NK cells display an immature phenotype when compared with peripheral blood NK cells, although this does not appear to diminish their functional capacity. A clinical trial using multiplex gene engineered iPSC derived CD19 CAR-NK cells, also expressing an IL-15/IL-15 receptor fusion protein supporting persistence, and a high affinity CD16 receptor to support enhanced ADCC (FT596) in combination with an anti-CD20 monoclonal antibody is underway. Given the versatility of this approach, early indicators of efficacy and safety are eagerly awaited. The effect of T-cell composition on CAR-T products is now well recognized. NK cells also display phenotypic and functional diversity with recognizable maturation stages potentially modulating the characteristics of CAR-NK products. While CAR expression has been shown to activate hypofunctional, uneducated and less mature NK cell subsets in vitro, relatively greater CAR-NK functional potential was observed for mature, adaptive and educated populations. Leveraging this effect, CIML NK cells modified to express a CD19 CAR showed synergism of CAR activation and CIML NK features in a model of relapsed lymphoma. Inhibitory NKG2A signaling, most relevant for immature NK cell subsets, does appear to be overcome by CAR mediated activation, however inhibitory KIR interactions were capable of dampening CAR-NK activation. Thus, the exact NK cell subset composition and KIR/HLA interactions warrant careful consideration in the development of CAR-NK products. CARs which are designed to signal with NK rather than T-cell based signaling domains may further tune the activation of CAR-NK cells, and this topic has been recently thoroughly reviewed. ## Aml target antigens and car-nk Despite the potential benefits of an NK cell approach, the pool of AML relevant CAR target antigens remains unchanged. It is also important to consider NK cell expression of potential targets to account for possible fratricide which could limit efficacy. Furthermore, differential sensitivity to NK cell innate and alloreactivity between the antigen negative populations for each target, could influence target choice using a CAR-NK approach. Most reports of CAR-NK cell activity at present describe pre-clinical findings. The status of CAR-NK therapies in development for prominent AML antigens is summarized in. CD33 expression has been documented in subsets of activated NK cells (acting as an inhibitory receptor) but sufficient to impair NK viability using a tri-specific immune-ligand molecule. NK cell CD33 knockout could thus serve a dual purpose of reducing fratricide and enhancing NK cell activation in a CD33 CAR-NK design. CD33 CAR NK-92 cells have been evaluated in a small phase I clinical trial by Tang et al.. While multiple infusions of CD33 CAR-NK-92 appeared safe following salvage chemotherapy in relapsed disease, it is not possible to infer anti-leukaemic effect in this study and the focus remains overcoming the limitation of irradiation. Recently, targeted gene insertion into a safeharbor locus via homologous repair using CRISPR/Cas9 gene editing in combination with adeno-associated virus (AAV)-mediated gene delivery was used to generate primary CD33 CAR-NK cells with confirmation of in vitro CD33 positive AML targeting. Several reports covering peripheral blood, cord blood and NK-92 cells expressing a CD123 CAR confirm in vitro activity, although Christodoulou et al. failed to see leukaemic control in a xenograft model. The latter observation was associated with poor CAR-NK persistence, highlighting a need for separate measures to enhance persistence beyond CAR expression. Our group and collaborators have recently reported the in vitro activity of affinity optimized CD38 CAR-NK cells in AML. CD38 is expressed on primary expanded NK cells, introducing a risk of NK cell fratricide, which can be overcome by CRISPR/Cas9 CD38 knockdown during manufacturing. Interestingly, CD38 knockdown in NK cells is associated with augmented rather than impaired activity, and resistance to oxidative stress relative to wild type NK cells. Although NK cells express the NKG2D receptor, expression of an NKG2D CAR enhances NK cell activity and should not be subject to downregulation encountered with endogenous NKG2D in AML. NKG2D CAR NK-92 and primary CAR-NK cells have been evaluated pre-clinically against multiple myeloma, and activated CAR-NK cells appeared to outperform CAR-T cells in one animal model. A phase I clinical trial of a haploidentical donor derived CAR-NK product targeting NKG2DL in MDS and AML has recently opened (NCT04623944). An existing clinical report of transient NKG2D CAR expression and local delivery leading to responses in colorectal cancer is notable as other examples which purposefully limit the duration of CAR expression have not proven successful. Local delivery is not feasible in AML but modifying the cells further to enhance homing may optimize the early activity of transiently expressed CARs. Dong et al. recently presented pre-clinical activity of CAR CIML-NK cells targeting NPM1c/HLA-A2. Relative to NPM1c CAR-T cells, it could be assumed that antigen escape via loss of HLA class I expression would be mitigated due to a concurrent enhancement of innate CIML-NK activity. Interestingly, not all off tumor activity can be considered negative when evaluating target antigens. CD33 and CD38 are expressed on MDSC populations, and NKGD2L may also mark Treg cells for elimination supporting a more NK cell favorable TME. ## Engineering persistence: a balance of efficacy and off-tumor effects The question of persistence applied to AML for CAR-T and CAR-NK cell therapies is more complex than for CD19 positive malignancies. The limitations in persistence of some CAR-NK cell designs, could be viewed as beneficial in limiting the duration of myelosuppressive effects for certain antigens. Accumulated experience in NK ACT and CAR-T cell therapies, suggests that this limitation in persistence may also compromise efficacy. The ideal balance remains to be defined, and may vary across target antigens, NK cell sources, and clinical scenarios. This concern does not apply to some targets already discussed (e.g., NKG2D and NPM1c CARs), which do not entail the same risk of offtarget effects, where long term CAR-NK persistence can be viewed as being a desirable characteristic. In other cases, and relevant for CAR-NK but not CAR-T applications, uncoupling the persistence of CAR signaling from haploidentical NK cell persistence could provide an ideal balance, given the established role of alloreactive NK cells in AML remission maintenance post ASCT. Optimized application of transient CAR expression, or inducible CAR/co-stimulatory signaling could realize this concept. Several means of supporting NK cell persistence have been reported which aim to avoid the need for exogenous non-targeted cytokine support. The MD Anderson cord blood CAR-NK process includes an IL-15 domain which provides for secretion and autocrine stimulation of NK cells without increasing circulating IL-15 levels when applied clinically. FATE therapeutics iPSC CAR-NK product FT596 includes a constituently active IL-15/IL-15 receptor fusion domain to provide autonomous signaling. Several groups have reported on the role of knocking out the cytokine-inducible sh2-containing protein (CISH) gene, a negative regulator of IL-15 signaling, enhancing NK cell metabolism, cytotoxicity, NK cell persistence and CAR-NK functionality. Notably, CISH knockout NK cells mediated improved disease control in an AML xenograft model. Wang et al. recently reported the benefit of inducible Myd88/CD40, providing co-stimulation as a freestanding protein separate to the CAR construct, enhancing persistence and cytotoxicity of CD123 CAR-NK cells. Myd88/CD40 represents a common downstream signaling molecule for cytokines known to support NK cell function. Notably, in this study, first generation CAR expression, autocrine IL-15 and Myd88/CD40 expression were required for tumor control in a xenograft model, and the authors suggest that separating these signals could also abrogate exhaustion in the long-term. CIML-NK cells display improved persistence in xenograft models, but the extent to which this translates to clinical applications continues to be explored. ## Next steps: optimizing car-nk activity in aml AML biology and clinical experiences with CAR-T and NK ACT infer a need to consider approaches augmenting CAR-NK cell therapies beyond antigen specificity. Gene transfer technologies, and pharmacological approaches to modulating NK cells, AML blasts and the TME are summarized in. We will consider the varied pathways to improving CAR-NK activity through NK cell homing capability, disrupting inhibitory checkpoints, modulating AML ligand expression, dual targeting, and NK cell persistence (previously discussed). ## Car-nk cell homing The density of NK cells on bone marrow core biopsies performed after NK ACT for AML correlates with clinical responses. Rapid homing of NK cells to bone marrow could allow for activity before in vivo suppressive factors act and increase the effective ratio of CAR-NK cells to targets. This aspect of CAR-NK design may be especially relevant for approaches that seek to leverage transient CAR expression to balance offtarget effects. Depending on the method, expression of the chemokine receptor CXCR4, as well as that of E-selectin ligands, can decrease during ex vivo NK cell expansion and the gain of function variant CXCR4 R334X has been shown to enhance NK cell homing to bone marrow when introduced prior to adoptive transfer in animal models. The tethering of circulating leukocytes to endothelium, the first step in leukocyte transmigration, is predominantly mediated by endothelial E-selectin binding of leukocyte E-selectin ligands (cell surface glycolipids and glycoproteins such as CD44, expressing sialyl Lewis X). Bone marrow vasculature constituently expresses E-selectin but this is upregulated in AML, an important mechanism by which leukemic cells (which also highly express E-selectin ligands) maintain their bone marrow niche and evade chemotherapy. The application of translational glycobiology to CAR-T engineering has recently been reviewed. Sialyl Lewis X expression can be enhanced on NK cells through fucosyltransferase activity, the key enzymatic step which determines sialyl Lewis X production in humans. This principle was shown to enhance bone marrow homing in a xenograft lymphoma model using NK-92 cells treated ex vivo with human Alpha-(1,3)-fucosyltransferase (Fut6) and GDP-fucose. ## Inhibitory checkpoints Inhibitory pathways represent potential NK cell checkpoints modulating CAR and innate signaling in NK cells. Genetic engineering could provide for robust knockdown of inhibitory receptors, although caution is warranted given the importance of certain inhibitory pathways in NK cell education and licensing, where a separate and carefully timed antibody-based receptor blockade may be preferable. Monoclonal antibodies to many of these targets have been developed and evaluated in clinical trials across a variety of hematological and solid organ malignancies, and several appear especially relevant to AML therapy. Despite promising pre-clinical findings a monoclonal antibody blocking the interaction between inhibitory KIRs (KIR2DL1/2/3) and HLA-C, lirilumab, failed to prolong leukemia free survival for elderly AML patients in remission as monotherapy, and notably triggered concern that continuous KIR inhibition could impair NK cell immune surveillance. The study group proposed that a secondary activating stimulus may be necessary to see benefit from KIR inhibition. In vitro data supports the concept that iKIR can dampen CAR signaling, although clinical exploration of this combination would require a careful dosing schedule. NK cell NKG2A is upregulated in AML, and a population of strongly NKG2A inhibited NK cells dominates recovery post haploidentical ASCT which has prompted investigation of the NKG2A monoclonal antibody monalizumab in this setting. Interestingly, in vitro data suggests CAR expression overcomes NKG2A negative regulation in NK cells, although an enhancement of innate signaling could still be an important contributor to overall efficacy. Elimination of surface NKG2A expression enhanced NK cell activity against primary AML blasts and no indications of NK cell impairment were seen in xenograft solid tumor models. AML is a disease with a relatively low mutational burden and poor responses to programmed cell death protein 1 (PD-1) blockade were encountered with monotherapy. Combination approaches and augmenting alloreactive T-cell responses post ASCT are now the focus of investigation. NK-cells and blast cells do express PD-1 and PD-L1, respectively and blockade using a PD-1 monoclonal antibody or scFv enhanced NK cell cytotoxicity against AML targets in vitro, although appears to be most relevant for resting rather than cytokine activated NK cells. Inhibition of TIGIT in AML is intuitive, however conflicting reports regarding its effect in experimental systems in vitro suggest caution is warranted. An antibody recognizing CD200 positive blast cells enhances the activity of cytokine induced killer cells, and the expression of CD200 on AML LSCs suggests this may be a useful pathway to target. Isolation of NK-92 cells negative for the NK inhibitory receptor Siglec-7 (which recognizes sialic acid containing siglec-ligands on the target cell surface), identified enhanced cytolytic activity against AML blasts -while disruption of the interaction of Siglec-7 with CD43 (a dominant source of Siglec-7 ligands) also enhanced NK cell anti-leukemic activity in vitro. Denosumab (a clinically available monoclonal antibody against RANKL) has been shown to overcome the immunosuppressive signaling associated with blast cell RANKL and interactions with NK cell RANK. ## Aml ligand expression and dual targeting Combining with treatments which modulate the expression of the CAR target antigen, or the NK cell receptor ligand profile of blast cells is another route to enhancing CAR-NK activity. Hypomethylating agents, ATRA and HDAC inhibitors modify NKG2D ligand expression in AML especially relevant to NKG2D CAR therapy but also antigen negative innate reactivity, while the CDK inhibitor dinaciclib enhances NK cell recognition of AML blasts predominantly by reducing blast cell HLA-E expression. Suppression of NKG2D ligands on LSCs specifically can be overcome by poly (ADP-ribose) polymerase 1 (PARP1) inhibitionPARP1 inhibition has also been shown to induce death receptor 5 (DR5) on AML blasts, sensitizing to killing by TRAIL. Immunomodulatory agents lenalidomide and pomalidomide enhanced NK cell cytolytic activity against AML via a heterogenous effect on NKG2D ligands and CD155 and a consistent decrease in HLA class I expression. ATRA also upregulates CD38 acting through a retinoic acid response element in the CD38 gene, and sensitizes KG1a cells and primary AML samples to CD38 CAR-NK activity. Recent evidence suggesting a negative effect of direct ATRA exposure on NK cell cytotoxicity warrants caution in the timing of this combination. While dual CAR targeting may increase the proportion of AML blasts eliminated by antigen specific recognition as discussed previously, some mechanisms of immune escape may still persist, including failure of the leukaemic cells to bind perforin and resistance to granzyme mediated cell death through leukemic expression of serpinB9/protease inhibitor-9. Here, an alternative mechanism of killing through death receptor pathways may be required. TRAIL is expressed by NK cells, and is considered a desirable feature of 'activation' in the context of NK cell expansion. Recent evidence suggests that death receptor mediated killing may be particularly important after pre-formed stores of perforin and granzyme are depleted, providing a backup mechanism of cytotoxicity. Ongoing 'serial' death receptor mediated killing also appears to be limited however, perhaps due to exhaustion of pre-formed death ligand, or degradation within the immune synapse during an initial killing event. NK cells can be engineered to constitutively express novel high affinity TRAIL variants with specificity for one or other TRAIL receptor, reducing the role of 'decoy' receptors and presenting a viable approach to enhanced death receptor mediated killing. The expression of these TRAIL in a membrane bound form induces optimal death receptor clustering, propagating a potent apoptotic stimulus. A DR4 variant TRAIL is under investigation to mitigate antigen escape in AML. Pharmacological approaches to modulating TRAIL receptor expression could also be applied-the proteosome inhibitor bortezomib is considered the most well-known, and clinically relevant TRAIL sensitizing agent via upregulation of DR5 expression. An element of caution is also required when engineering NK cell death ligand expression-acknowledging the role that death receptor mediated killing has in modulating immune responses and the potential for fratricide. ## Car-nk in the context of aml immunotherapy At the essence of CAR-NK cell therapies is the conferral of antigen specific targeting to alloreactive NK cells, augmenting their innate anti-leukaemic activity. The overlapping fields of CAR-T and NK ACT have been considered above, however several other prominent and related approaches to AML immunotherapy are in development. Cytokine administration has been investigated to enhance endogenous immunity against AML. IL-2 in combination with histamine dichloride improved LFS in a phase III trial as a consolidation therapy likely through recruiting NK cells, but has not been widely adopted into clinical practice. The IL-15 superagonist ALT-803 achieved responses in patients with relapse post ASCT, and may be combined with other NK cell directed therapies or ACT in the future. Unconjugated antibodies alone have not proven beneficial in AML. The ADC gemtuzumab ozogomycin improves overall survival when combined with induction chemotherapy in CD33 positive favorable and intermediate risk AML. ADCs against other targets are in development. Bispecific and trispecific antibody constructs engage immune cells and targets through simultaneous recognition of a cancer antigen and immune cell surface proteins. This principle has been explored for NKG2DL targeting using an NKG2D-CD16 fusion protein. Trispecific killer engager (TriKE) constructs include an IL-15 moiety to stimulate as well as recruit NK cells, and are in development targeting CD33 and CLL-1. These novel agents recruit endogenous NK cells in an antigen specific manner. Relative logistical simplicity is a potential advantage over CAR-NK, although whether the inherent dysfunction of NK cells in active AML will be sufficient to constrain this elegant approach remains an active question, and the heterogenous expression of these antigens is equally relevant here. Application of these agents as post remission therapy with restored NK cell function or indeed combination with NK cell adoptive transfer which would also permit alloreactivity may ultimately be considered. T cells with engineered TCRs can target a range of intracellular antigens including both TAAs and neoantigens. This bypasses the heterogeneity in cell surface antigens. The TAA Wilms tumor 1 (WT1) has been investigated as a target of both a high affinity engineered TCR-T cell therapy and a vaccine based approach post ASCT with favorable outcomes. # Conclusions CAR-NK cell therapies are at an early stage of investigation in AML, but present an appealing combination of antigen specific, innate and alloreactive activity supported by engineered persistence. The closely related fields of CAR-T cell therapy and NK cell ACT have provided a wealth of information which can be applied to expedite the development of this platform, although challenges remain. The range of NK cell sources, expansion methods, CAR designs and combination approaches offer a toolbox with which to tackle the inherent complexity of AML treatment. If the potential of CAR-NK is realized, this approach may strike an ideal balance in the challenging but expanding field of AML immunotherapy and ultimately improve treatment responses while reducing toxicities.

Incidence of Adverse Drug Reactions in COVID‐19 Patients in China: An Active Monitoring Study by Hospital Pharmacovigilance System ## Incidence of adverse drug reactions in covid-19 patients in china: an active monitoring study by hospital pharmacovigilance system Ji Sun 1 , Xuanyu Deng 1 , Xiaoping Chen 2,3,4 , Juanjuan Huang 1 , Siqiong Huang 1 , Yanfei Li 1 , Jinhui Feng 1 , Jiyang Liu 1, * and Gefei He 1, * To evaluate the incidence, type, and risk factors associated with adverse drug reactions (ADRs) among patients with coronavirus disease 2019 (COVID-19) by Hospital Pharmacovigilance System (CHPS). A retrospective analysis was performed on 217 patients with COVID-19 admitted to the First Hospital of Changsha in China, from January 17, 2020, to February 29, 2020. The active monitoring model in CHPS was used to detect ADR signals of the hospital information system. The risk factors for the ADRs were classified using the World Health Organization-Uppsala Monitoring Centre (WHO-UMC) system. Univariate and multivariate logistic regressions were carried out to analyze the risk factors of ADRs. Our results showed that the prevalence of ADRs was 37.8% in the patients, which was predominated by drug-induced gastrointestinal disorders and liver system disorders (23.0% vs. 13.8%). The ADR could be explained by the use of lopinavir/ ritonavir and umifenovir by 63.8% and 18.1%, respectively. There were 96.8% of ADRs that occurred within 14 days of hospitalization. Multivariable analysis showed that length of stay (odds ratio (OR): 2.02; 95% confidence interval (CI) 1.03-3.96; P = 0.04), number of drugs used in the hospital (OR: 3.17; 95% CI 1.60-6.27; P = 0.001) and underlying basic diseases (OR: 2.07; 95% CI 1.02-4.23; P = 0.04) were independent risk factor for ADRs in the patients. Together, the incidence of ADRs was significantly high during the treatment period. Moreover, the active monitoring of the CHPS system reflected ADRs during COVID-19 treatment in the real world, which provided reference for safe medication in the clinic. In late December 2019, a cluster of cases with severe acute respiratory syndrome 2019 novel coronavirus pneumonia (SARS-CoV-2) infection in Wuhan, China, aroused worldwide concern. 1 As of March 9, 2020, over 100 countries reported laboratory-confirmed cases of coronavirus disease 2019 (COVID- [bib_ref] Assessment of the pharmacokinetic interaction between eltrombopag and lopinavir-ritonavir in healthy adult..., Wire [/bib_ref] , and > 110,000 cases have been diagnosed, with an estimated mortality risk of ~ 3%.Unfortunately, no drug or vaccine has yet been approved to treat SARS-CoV-2 infection.Lopinavir/ritonavir, interferon, umifenovir, chloroquine, remdesivir, favipiravir, anti-inflammatory drugs (such as corticosteroids and other molecules), Chinese traditional medicines, such as ShuFeng JieDu capsules and Lianhuaqingwen capsules, Xuebijing injections are potential options for the treatment of novel CoV. [bib_ref] Drug treatment options for the 2019-new coronavirus (2019-nCoV), Lu [/bib_ref] [bib_ref] Favipiravir: pharmacokinetics and concerns about clinical trials for 2019-nCoV infection, Du [/bib_ref] However, the treatment scheme is still in the exploratory stage. A report [bib_ref] Pharmacological care strategy for antivirals in patients with COVID-19 complicated by underlying..., Wang [/bib_ref] showed that the treatments were complex in co-combination patients with COVID-19 with basic diseases. The risk for drug-related adverse reactions is increased. At present, the recommended drugs, such as HIV protease inhibitors, have complex interactions, 7 interferon usage is special, [bib_ref] Compounds with therapeutic potential against novel respiratory 2019 coronavirus, Martinez [/bib_ref] ribavirin has relatively more adverse reactions, [bib_ref] Identification of severe acute respiratory syndrome in Canada, Poutanen [/bib_ref] wheres no evidence is available for the use of umifenovir, 10 chloroquine, [bib_ref] Breakthrough: chloroquine phosphate has shown apparent efficacy in treatment of COVID-19 associated..., Gao [/bib_ref] and remdesivir. [bib_ref] First case of 2019 novel coronavirus in the United States, Holshue [/bib_ref] Therefore, drug safety cannot be ignored while ensuring efficacy. Adverse drug reactions (ADRs) range from mild to life-threatening with short-term and long-term effects. However, little is known about the incidence of ADRs in patients with COVID-19. The China Hospital Pharmacovigilance System (CHPS) [bib_ref] Active pharmacovigilance in China: recent development and future perspectives, Li [/bib_ref] was developed by China National Center for Adverse Drug Reaction Monitoring (CNCAM) to collect and analyze information automatically extracted from sentinel hospitals. It may partially solve the problems of under-reporting, undue delays, and miscommunication. Better reporting systems can also have a positive effect on rational drug use in medical institutions. The real drug safety data were extracted to provide decision-making basis for drug risk management. In this study, to investigate the ADRs of the patients in the real world, we used the CHPS system to actively monitor the medication safety of patients with COVID-19, which provide reference for clinically safe medication. # Methods ## Study design and population A retrospective study was carried out in this study. Two hundred seventeen patients with COVID-19 who were transferred from the hospitals in Changsha to the First Hospital of Changsha, the designated hospital, from January 17 to February 29, 2020, were enrolled. Cases of COVID-19 were diagnosed according to the World Health Organization (WHO) interim guidance for all the patients. The study was approved by Ethics Committee of the First Hospital of Changsha and written informed consent was waived because of the retrospective nature of the study. All patients were codified and anonymized to protect the confidentiality of individual participants. After data coding and analysis, all records were deleted to further protect participants' confidentiality. Active monitoring All treatments used in the patients with COVID-19 were selected in the study, then through the literature, instructions of the US Food and Drug Administration (FDA), and global trigger tool white paper to establish the trigger items for depression in patients [fig_ref] Table 1: The trigger items for active monitoring of ADR in inpatients with COVID-19 [/fig_ref]. A retrospective chart review of a sample of 217 patients with COVID-19 transferred from the hospitals in Changsha to the designated hospital from January 17 to February 29, 2020, was carried out. All the medical records with identified triggers to determine the presence of ADRs by the active monitoring model in CHPS were used. Two clinical pharmacists checked the medical records of the system alarm cases one by one for relevance evaluation. ## Article ## Data collection The characteristics of the patients with COVID-19 (time of admission, length of stay, sex, age, basic diseases, etc.), history of drug allergies, the Antiviral Protocol, and the number of medications used during hospitalization were extracted. ADRs were evaluated when the trigger was positive. Then, the causality, the time of occurrence, suspicious drugs, and clinical outcome of ADRs were recorded. ## Case assessment Causality assessment was performed for all suspected ADRs using the WHO-Uppsala Monitoring Centre (WHO-UMC) system. 14 The WHO-UMC system is a universally accepted method for causality assessment. [bib_ref] Causality assessment: a brief insight into practices in pharmaceutical industry, Naidu [/bib_ref] The relationship between the reported ADRs and drugs was categorized as certain, probable, possible, unlikely, conditional/unclassified, or unassessable/unclassifiable. Only cases categorized as certain, probable, and possible were included. Seriousness of the identified suspected ADRs was determined according to the definition of the International Conference on Harmonization (ICH) E2A guideline (ICH E2A Clinical Safety Data Management: Definitions and Standards for Expedited Reporting).According to the ICH E2A guideline, a serious adverse event (AE) or reaction is any untoward medical occurrence that at any dose: Resulted in death, Is life-threatening, Required hospitalization or resulted in prolongation of existing hospitalization, Resulted in persistent or significant disability/incapacity, or Caused congenital anomaly/birth defect or medically important event or reaction that required medical/surgical intervention to prevent serious outcome. The clinical outcome indicators of ADRs generally include death, cured, improvement, recovered with sequelae, no healing, and unknown. The clinical cure of an ADR was considered when ADR symptoms disappeared or recovery of the abnormal indexes to normal values was observed. Data processing and statistical analysis Data were captured into a computer using an entry program developed with the WPS software package. Data were edited during and after data entry using WPS and SPSS 20. Both descriptive and analytical analyses were carried out on the data using SPSS. Both univariate and multivariate analyses were carried out to evaluate the associations of potential risk factors with the risk for ADRs. Results were presented as percentages and frequencies as appropriate. To test statistical significance, 95% confidence intervals (CIs) and/or P values were used. A P value < 0.05 was regarded as being statistically significant. # Results ## Characteristics of patients Two hundred seventeen patients were included in this study [fig_ref] Table 2: Characteristics of patient between those with ADRs and without ADRs Severe COVID-19... [/fig_ref]. Of the 217 patients, 111 (51.2%) were women and 106 (48.8%) were men. The mean age was 45.7 ± 16.6 years. There were 28.6% of them who had underlying basic diseases (hypertension, cardiovascular disease, cerebrovascular disease, diabetes, cancer, chronic kidney disease, chronic liver disease, HIV, and chronic obstructive pulmonary disease). Of the 217 patients, 118 patients showed positive triggers, and 36 patients were excluded because the positive trigger was due to [bib_ref] Causality assessment: a brief insight into practices in pharmaceutical industry, Naidu [/bib_ref].9 ± 7.52; P < 0.001) was significantly longer in the ADRs group. The number of drugs used in the hospital (8.57 ± 3.34 vs. 5.40 ± 2.10; P < 0.001), history of drug allergies (3 (2.2%) vs. 7 (8.5%); P < 0.044), and combined use of antiviral agents (95 (70.4%) vs. 68 (82.9%); P < 0.044) were significantly higher in the ADRs group. There were no significant differences in the proportions of sex and underlying basic diseases between the two groups. ## Risk factors for adrs in patients with covid-19 To identify risk factors associated with the occurrence of ADRs for the patients with COVID-19, a retrospective study was conducted. Univariate analysis showed that the following factors were extensively associated with the occurrence of ADRs: length of stay, combined use of antiviral agents, number of drugs used in the hospital, and history of drug allergies. The results of the multivariate analysis were shown in [fig_ref] Table 3: Univariate and multivariate analysis of risk factor for ADRs in patients with... [/fig_ref] : the independent risk factors for the occurrence of ADRs in patients with COVID-19 were length of stay (odds ratio (OR): 2.02; 95% CI 1.03-3.96; P = 0.04), number of drugs used in the hospital (OR: 3.12; 95% CI 1.60-6.27; P = 0.001) and underlying basic diseases (OR: 2.07; 95% CI 1.02-4.23; P = 0.045). ## Characteristics of adrs Drug-related adverse reactions, as categorized by the system used, and the onset times are listed in [fig_ref] Table 4: Involved organs and systems of ADRs for the patients with COVID-19 [/fig_ref] , respectively. Gastrointestinal (GI) disorders (23.0%) were the most frequent ADRs, followed by liver disorders (13.8%), skin and appendages disorders (4.15%), and hyperlipemia (1.38%). Three cases of hyperlipidemia were caused by lopinavir/ritonavir. One case was a 37-year-old male patient with normal blood lipids before admission. On February 9, he was given lopinavir/ ritonavir. The triglyceride value was 6.35 and 12.0 mmol/L on February 10 and 14, respectively. On February 14, the patient stopped using lopinavir/ritonavir. On February 19, the triglyceride was 10.3 mmol/L. Therefore, the patient was given fenofibrate capsules on the same day. The triglyceride decreased to 4.62 mmol/L until February 22. It was worth noting that two of the three cases of hyperlipidemia were in children. In terms of time of onset of the ADRs, 9.57% (rash, diarrhea, and vomiting) occurred within the first day of treatment, 79.8% occurred within 7 days of treatment, and 96.8% occurred within 14 days of treatment. Suspected drugs with ADRs are listed in [fig_ref] Table 6: Suspicious drugs, casualty assessment, and prognosis of ADRs for the patients with... [/fig_ref] and [fig_ref] Table 1: The trigger items for active monitoring of ADR in inpatients with COVID-19 [/fig_ref]. Most of the reactions were associated with lopinavir/ritonavir and umifenovir with 63.8% and 18.1%, respectively. Chloroquine and antibacterial drugs were similar in causing ADRs with incidence of 5.31% and 4.25%, respectively. Of these, only 6.38% were found to be serious. The causal relationship assessed by using the WHO-UMC system in the suspected ADR cases were found to be probable and possible (55.3% vs. 44.7%). The prognosis of ADRs in these patients were favorable, with 62.8% cured and 37.2% improved. # Discussion Few studies regarding drug safety monitoring are reported in patients with COVID-19. Only one study [bib_ref] Retrospective analysis of diarrhea associated with lopinavir-ritonavir for COVID-19 and its prevention, Yang [/bib_ref] in China reported diarrhea associated with lopinavir/ritonavir use in patients with COVID-19. The study showed that after using lopinavir/ritonavir in 33 patients with COVID-19 in the Nanchong area in China, 15 patients had diarrhea, rash, and other ADRs, the incidence was 42.9%. In our study, we used the CHPS system to actively monitor the drug safety issues of patients with COVID-19 for the first time. This ADR monitoring system has improved work efficiency and the frequency of ADR reporting. It may partially solve the problem of under-reporting, undue delays, and miscommunication, and better reflect the ADRs for patients with COVID-19 in the real world. [bib_ref] Active pharmacovigilance in China: recent development and future perspectives, Li [/bib_ref] Our study showed that ADRs in patients with COVID-19 were mainly characterized by GI reactions, liver injury, rash, and hyperlipidemia, with incidence of 23.0%, 13.8%, 4.15%, and 1.38%, respectively. There were 96.8% of ADRs that occurred within 14 days. GI reactions occurred earlier, usually within 7 days. The proportion of ADRs (63.8%) caused by lopinavir/ritonavir was highest. The incidence of ADRs by lopinavir/ritonavir was 33.5% (60/179), which was lower than the Yang et al. study. [bib_ref] Retrospective analysis of diarrhea associated with lopinavir-ritonavir for COVID-19 and its prevention, Yang [/bib_ref] The difference might be explained by the small sample size in the Yang et al. study. [bib_ref] Retrospective analysis of diarrhea associated with lopinavir-ritonavir for COVID-19 and its prevention, Yang [/bib_ref] The results of other studies are slightly different, which may be related to the inconsistency of the study population. The safety monitoring data of other studies are mainly concentrated in patients with AIDS and healthy people. [bib_ref] Short communication: comparable safety and efficacy with once-daily versus twice-daily dosing of..., Gonzalez-Garcia [/bib_ref] [bib_ref] Assessment of the pharmacokinetic interaction between eltrombopag and lopinavir-ritonavir in healthy adult..., Wire [/bib_ref] For example, as seen in the Gonzalez-Garcia et al. study, the most frequently reported AE in healthy adult subjects was diarrhea (26/40; 65%). [bib_ref] Short communication: comparable safety and efficacy with once-daily versus twice-daily dosing of..., Gonzalez-Garcia [/bib_ref] A randomized trial to evaluate the efficacy and safety of lopinavir/ritonavir in the treatment of HIV-1 infection reported that the incidence of diarrhea in moderate/severe AEs was 16%. [bib_ref] Assessment of the pharmacokinetic interaction between eltrombopag and lopinavir-ritonavir in healthy adult..., Wire [/bib_ref] The incidence of adverse reactions by umifenovir and chloroquine were 17 of 119, 14.3% vs. 5/37, 13.5% respectively, mainly manifested as GI reaction and liver injury. Decrease in vision or cardiotoxicity was not observed in patients using chloroquine. [bib_ref] Revisiting the cardiotoxic effect of chloroquine, Blignaut [/bib_ref] [bib_ref] Variability of chloroquine and hydroxychloroquine retinopathy among various ethnicities, Giocanti-Auregan [/bib_ref] This is because chloroquine induces visual toxicity that is generally associated with long-term use. However, the duration of treatment by chloroquine in patients with COVID-19 was generally < 10 days in our study. After symptomatic support treatment, all the ADRs turned out well, 62.8% were cured, and 37.2% were improved. In all the ADRs Hypertriglyceridemia and hypercholesterolemia were the most frequently observed laboratory abnormalities in lopinavir/ritonavir recipients in clinical trials and may be the reason for discontinuation of therapy in some patients. [bib_ref] Predictive factors of lopinavir/ritonavir discontinuation for drug-related toxicity: results from a cohort..., Bongiovanni [/bib_ref] Increases in total cholesterol and triglycerides were seen within the first month of starting therapy and were relatively stable then after. [bib_ref] Lopinavir/ritonavir in the treatment of HIV-1 infection: a review, Chandwani [/bib_ref] In our study, three cases of hyperlipidemia caused by lopinavir/ritonavir were noted. It is worth noting that two of the three cases of hyperlipidemia were in children. In the process of clinical use, it is necessary to closely monitor the changes of blood lipids in pediatric patients. Because ADRs are the single most common reason for poor adherence to treatment, identification of risk factors for the occurrence of ADRs is essential to optimize the initial choice of antiretroviral regimen before initiating therapy and to adapt the pace of surveillance to each unique situation. Our study showed that the independent risk factors for the occurrence of ADRs in patients with COVID-19 included length of stay (OR: 2.02; 95% CI 1.03-3.96; P = 0.04), number of drugs used in the hospital (OR: 3.17; 95% CI 1.60-6.27; P = 0.001), and underlying basic diseases (OR: 2.07; 95% CI 1.02-4.23; P = 0.045). One study observed that the ADR was significantly associated with the number of drugs and urgent hospital admission. [bib_ref] Risk factors for adverse drug reactions in older inpatients of geriatric wards..., Kojima [/bib_ref] However, most patients were admitted through outpatient service in our study, so the factor of urgent hospital admission was not involved. Polypharmacy has been reported to be a strong risk factor for ADR in several studys.The length of stay was reported to be significantly associated with the occurrence of ADRs in univariate analysis in the Kojima et al. study. [bib_ref] Risk factors for adverse drug reactions in older inpatients of geriatric wards..., Kojima [/bib_ref] In addition, there is also significant association between the age and the occurrence of ADR, but this was not observed in our study. Although other studies have associated age as a risk factor for ADR, [bib_ref] Prevalence, risk factors and main features of adverse drug reactions leading to..., Pedros [/bib_ref] our study did not show any association between age and ADRs. The interpretation of these results is limited to the context from which participants were drawn. The randomized controlled trial cohort consisted of patients from health facilities in Hunan Province and may not be representative of patients across China. In addition, partial evaluation of possible ADR does not completely exclude the influence of disease. However, in our study, we try our best to avoid the interference caused by the COVID-19 infection. Patients with abnormal indicators or related symptoms and diseases before admission cannot be judged as an ADR according to the evaluation criteria of ADRs. Furthermore, we only observed adverse reactions during hospitalization. However, late-cut retinopathy caused by chloroquine may occur many years after discontinuation of the treatment. So further studies with more extended follow-up periods are needed to assess the longer-term implications of AEs and the potential fluidity in predictors of such events. ## Supporting information supplementary information accompanies this paper on the clinical Pharmacology & Therapeutics website (www.cpt-journal.com). [table] Table 1: The trigger items for active monitoring of ADR in inpatients with COVID-19 [/table] [table] Table 2: Characteristics of patient between those with ADRs and without ADRs Severe COVID-19 was mainly defined according to the Diagnosis and Treatment of Novel Coronavirus Pneumonia (revised version fifth) by the General Office of The National Health Commission of People's Republic of China, Office of National Administration. Patients with severe COVID-19 refer to patients with clinical classification of severe and critical types. The mean age of the patients without ADRs (No ADR) group and with ADRs groups was 46.0 ± 16.2 and 45.2 ± 17.5 years, respectively. Compared with the No ADRs group, the length of stay (21.1 ± 8.63 vs. [/table] [table] Table 3: Univariate and multivariate analysis of risk factor for ADRs in patients with COVID-19 [/table] [table] Table 4: Involved organs and systems of ADRs for the patients with COVID-19 [/table] [table] Table 5: Frequency of ADRs for the patients with COVID-19 according to the time of onset ARTICLE observed, serious adverse reactions accounted for 18.1%, mainly liver injury and hyperlipidemia. For the 17 patients with serious adverse reactions, 14 had liver injury and 3 had hyperlipidemia. [/table] [table] Table 6: Suspicious drugs, casualty assessment, and prognosis of ADRs for the patients with COVID-19Data are n (%). Frequency was calculated as number/94*100%. ADRs, adverse drug reactions; COVID-19, coronavirus disease 2019; SADRs, serious adverse drug reactions. [/table]

A Rare Association of Congenital Diaphragmatic Hernia with Lower Esophageal Atresia and Perforation Congenital diaphragmatic hernia is known to be associated with esophageal atresia, which is a rare association. We report a rare occurrence of congenital diaphragmatic hernia and lower esophageal atresia. # Introduction Abnormalities of the esophagus such as gastroesophageal reflux disease (GERD), esophageal motility disorders, esophageal duplications cysts, and tracheoesophageal fistula with esophageal atresia are rare but documented occurrences with congenital diaphragmatic hernia [bib_ref] Abnormal esophageal anatomy associated with a congenital diaphragmatic hernia: report of a..., Karnak [/bib_ref] [bib_ref] Anatomic and functional abnormalities of the esophagus in infants surviving congenital diaphragmatic..., Stolar [/bib_ref]. We report a rare association of lower esophageal atresia with congenital diaphragmatic hernia (CDH). ## Case report A 10-day male child presented with respiratory distress without cyanosis since birth. On clinical examination, patient had tachypnea, with a scaphoid abdomen. The persistent drooling of saliva led to the suspicion of esophageal atresia. A red rubber catheter was passed into the esophagus, but there was resistance at 15 cm from the alveolar margin. Chest X-ray showed evidence of left CDH with mediastinal shift and the tip of nasogastric tube at the level of the diaphragm. A contrast esophagogram was done which showed holdup of dye at the level of the diaphragm [fig_ref] Figure 1: Contrast esophagogram showing holding up [/fig_ref]. With the suspicion of associated esophageal obstruction and CDH, a laparotomy was done using a chevron incision. The operative findings include left posterolateral CDH, complete disruption of the esophagogastric junction with a blindending esophagus, and a sealed esophageal perforation at the esophagogastric junction [fig_ref] Figure 2: Operative photograph showing a blind ending of the lower esophagus with a... [/fig_ref]. The surgical correction included repair of CDH and esophagogastric anastomosis with a feeding jejunostomy. The postoperative course was uneventful. Jejunostomy feeds were started on the 4th postoperative day. Contrast esophagogram was done on the 10th postoperative day. It showed free flow of dye into the stomach. At discharge, the child was on full oral feeds. He has been followed up for 3 months. The child's general condition is good with adequate weight gain. # Discussion Esophageal anomalies are known to be associated with CDH. These associations include tracheoesophageal fistula with esophageal atresia, GERD, esophageal dysmotility, esophageal duplication cysts, and esophageal ectasia [bib_ref] Abnormal esophageal anatomy associated with a congenital diaphragmatic hernia: report of a..., Karnak [/bib_ref] [bib_ref] Anatomic and functional abnormalities of the esophagus in infants surviving congenital diaphragmatic..., Stolar [/bib_ref]. The possible noted explanations include the kinking of the esophagogastric junction [bib_ref] Anatomic and functional abnormalities of the esophagus in infants surviving congenital diaphragmatic..., Stolar [/bib_ref] and abnormal innervation of the esophagus by the vagus and recurrent laryngeal nerve [bib_ref] The vagus and recurrent laryngeal nerves in experimental congenital diaphragmatic hernia, Martínez [/bib_ref]. Tracheoesophageal fistula with esophageal atresia is a known association of CDH, albeit a rare one with an incidence of 0.005 per 1000 births [bib_ref] Left diaphragmatic hernia associated with lower esophageal atresia, Takehara [/bib_ref]. In this case report, there is atresia of the lower esophagus at the esophagogastric junction. It appears to be an acquired event probably due to vascular compromise by the acute kinking of the stomach in the thorax because of the CDH. In an extensive review of the English literature, there is only one similar case reported International Journal of Pediatrics by van Dooren et al. [bib_ref] The cooccurrence of congenital diaphragmatic hernia, esophageal atresia/tracheoesophageal fistula, and lung hypoplasia, Van Dooren [/bib_ref] , where there was an atresia of the lower end of the esophagus that was not suspected until the postoperative period. In this case report, there was preoperative diagnosis of a lower esophageal obstruction; hence surgery was done using a chevron (rooftop) incision. This incision allows access for correction of both of the lesions. A feeding jejunostomy is a useful adjunct until the restoration of the esophagogastric continuity. In conclusion, esophageal anomalies are known to occur with CDH. Lower esophageal atresia is a rare association and a preoperative suspicion of this association, clinically, leads to timely correction of both of the lesions. A cheveron incision gives good access to correct both the CDH and lower esophageal atresia. ## Abbreviations CDH: Congenital diaphragmatic hernia GERD: Gastroesophageal reflux disease. [fig] Figure 1: Contrast esophagogram showing holding up (arrow) of contrast at the level of the diaphragm with evidence of CDH with lower esophageal obstruction. [/fig] [fig] Figure 2: Operative photograph showing a blind ending of the lower esophagus with a sealed perforation (arrow). [/fig]

Infantile systemic juvenile xanthogranuloma case with massive liver infiltration Infantile systemic juvenile xanthogranuloma (ISJXG) is an uncommon form of juvenile xanthogranuloma, a non-Langerhans cell proliferation of infancy and early childhood. In a small percentage of patients, the visceral involvement-most commonly to the central nervous system, liver, spleen, or lungs-may be associated with severe morbidity, and eventually fatal outcome. Here we describe the clinical and pathological findings of a 28-day-old girl with ISJXG who died with respiratory distress syndrome. She had few cutaneous lesions but massive liver and spleen infiltration; other affected organs were multiple lymph nodes, thoracic parasympathetic nodule, pleura, pancreas, and kidneys. Additional findings were mild pulmonary hypoplasia and bacteremia. Immunohistochemistry on fixed tissues is the standard for diagnosis. Immunophenotype cells express CD14, CD68, CD163, Factor XIIIa, Stabilin-1, and fascin; S100 was positive in less than 20% of the cases; CD1a and langerin were negative. No consistent cytogenetic or molecular genetic defect has been identified. This case demonstrates that the autopsy is a handy tool, because hepatic infiltration, which was not considered clinically, determined a restrictive respiratory impairment. In our opinion, this was the direct cause of death. # Introduction Dendritic and histiocytic neoplasms are rare, and together make up less than 1% of neoplasms presenting in the lymph nodes or soft tissues. [bib_ref] Other histiocytic and dendritic cell neoplasms, Chang [/bib_ref] These tumors are usually classified into two main groups based on their derivation from either mesenchymal cells or bone marrow precursors. [bib_ref] Dendritic cell and histiocytic neoplasms: biology, diagnosis, and treatment, Dalia [/bib_ref] [bib_ref] Clinicopathologic characteristics and outcomes of histiocytic and dendritic cell neoplasms: the Moffitt..., Dalia [/bib_ref] They occur less often during the perinatal period, and there is a study suggesting that there has been an increased incidence of spontaneous regression of certain histiocytic lesions in neonates compared to older individuals.Infantile systemic juvenile xanthogranuloma (ISJXG), an uncommon non-Langerhans cell proliferation of infancy and early childhood, 5,6 is one of the many clinical variants, with a common histopathology of the juvenile xanthogranuloma (JXG) family disorders namely (i) solitary or multiple dermal; (ii) infantile systemic; (iii) deep (soft tissue, "giant"); (iv) xanthoma disseminatum; (v) papular/generalized eruptive; (vi) benign cephalic histiocytosis; (vii) adult orbital XG; (viii) progressive nodular histiocytosis; and (ix) Erdheim-Chester disease. [bib_ref] The confusing state of the histiocytosis, Chu [/bib_ref] [bib_ref] Non-Langerhans cell histiocytosis: a new unifying concept, Zelger [/bib_ref] [bib_ref] Lyon: International Agency for Research on Cancer, Jaffe [/bib_ref] [bib_ref] Juvenile xanthogranuloma in childhood and adolescence: a clinicopathologic study of 129 patients..., Janssen [/bib_ref] Over the last decade, systemic forms have been increasingly reported.Stromal dendritic cells have been proposed (i.e., mesenchymal in origin) as the cells of origin, based on immunoreactivity to fascin and factor XIIIa. Immunohistochemistry, on fixed tissues, is the standard for diagnosis, and a panel of antibodies is required due to a lack of a unique cell marker. [bib_ref] The confusing state of the histiocytosis, Chu [/bib_ref] [bib_ref] Non-Langerhans cell histiocytosis: a new unifying concept, Zelger [/bib_ref] [bib_ref] Lyon: International Agency for Research on Cancer, Jaffe [/bib_ref] [bib_ref] Juvenile xanthogranuloma in childhood and adolescence: a clinicopathologic study of 129 patients..., Janssen [/bib_ref] Although the biology of ISJXG is not clear, it often presents spontaneous regression over several years,as in the isolated cutaneous form, the reason why the removal of these benign lesions is dissuaded. However, prompt and accurate diagnosis is essential. In a small percentage of patients with visceral involvementmost commonly in the central nervous system (CNS), liver, spleen, or lungs-ISJXG may be associated with severe morbidity. [bib_ref] Juvenile xanthogranuloma: forms of systemic disease and their clinical implications, Freyer [/bib_ref] [bib_ref] Successful therapy of systemic xanthogranuloma in a child, Unuvar [/bib_ref] [bib_ref] Disseminated juvenile xanthogranulomatosis in a newborn resulting in liver transplantation, Haughton [/bib_ref] [bib_ref] Severe congenital systemic juvenile xanthogranuloma in monozygotic twins, Chantorn [/bib_ref] [bib_ref] A rare case of neonatal systemic xanthogranulomatosis with severe hepatic disease and..., Papadakis [/bib_ref] Surgery, chemotherapy, and radiotherapy have been used with favorable results. [bib_ref] Juvenile xanthogranuloma: forms of systemic disease and their clinical implications, Freyer [/bib_ref] However, occasionally fatal outcome may occur. [bib_ref] Juvenile xanthogranuloma: forms of systemic disease and their clinical implications, Freyer [/bib_ref] [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] In a PubMed search, we found five reports of congenital fatal cases of ISJXG. [bib_ref] Juvenile xanthogranuloma in childhood and adolescence: a clinicopathologic study of 129 patients..., Janssen [/bib_ref] [bib_ref] Severe congenital systemic juvenile xanthogranuloma in monozygotic twins, Chantorn [/bib_ref] [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] [bib_ref] Fatal congenital systemic juvenile xanthogranuloma with liver failure, Hu [/bib_ref] [bib_ref] Systemic juvenile xanthogranuloma with fatal outcome, Azorín [/bib_ref] [bib_ref] Congenital disseminated juvenile xanthogranuloma with unusual skin presentation and renal involvement, Kolivras [/bib_ref] [bib_ref] Congenital systemic juvenile xanthogranuloma with placental lesion, Takeuchi [/bib_ref] [bib_ref] Prophylactic recombinant factor VIIa administration to an infant with congenital systemic juvenile..., Santiago [/bib_ref] [bib_ref] Deep-seated congenital juvenile xanthogranuloma: report of a case with emphasis on cytologic..., Barroca [/bib_ref] [bib_ref] Fatal congenital systemic juvenile xanthogranuloma with liver failure, Hu [/bib_ref] [bib_ref] Successful treatment of congenital systemic juvenile xanthogranuloma with Langerhans cell histiocytosis-based chemotherapy, Nakatani [/bib_ref] Herein we describe the clinical and pathological findings of a 28-day-old girl with congenital ISJXG who died with respiratory distress syndrome. In our opinion, the massive hepatomegaly caused lung restriction (which could have been present since the late gestation) hampering the pulmonary development, resulting in mild pulmonary hypoplasia. She had few cutaneous lesions but massive liver and spleen infiltration; other affected organs were multiple lymph nodes, the thoracic parasympathetic nodule, the pleura, the pancreas, and the kidneys. Additional findings were hypoplastic lungs and bacteremia. ## Case report This is the case of a 28-day-old girl without a remarkable family history, who was delivered at term after an uneventful pregnancy. She was born with counted bluish palpable nodular lesions from 0.5 to 1 cm, which were initially located on the face. At the age of 21 days, she was presented to a general hospital with irritability and progressive abdominal distention. On admission, she was pale and presented 13 visible and 6 palpable skin nodules located on her face, thorax, and extremities, and massive hepatomegaly and splenomegaly. Blood tests showed mild anemia and thrombocytopenia. The abdominal echography revealed multiple nodular lesions throughout the enlarged liver. After 24 hours, the mother decided to take her to a tertiary care hospital, where clinically congenital leukemia was suspected. The anemia and thrombocytopenia worsened, but leukocytes were normal. Serologic studies for HIV, cytomegalovirus, Epstein-Barr virus and Toxoplasma were negative. Serum tumor markers as alpha-fetoprotein, human chorionic gonadotropin, and carcinoembryonic antigen were negative. The platelet count ranged from 4,000 to 18,000/ mm 3 (reference range [RR]; 142-424 x 10 3 /mm 3 ), hemoglobin range was 7.5-11.3 g/dL (RR;12.2-18.1 g/dL); peripheral blood leukocyte count range was 4,580-8,520/mm 3 (RR; 4.60-10.20 x 10 3 /mm 3 ). Electrolytes showed persistent hyponatremia (sodium range 124-133 mEq/L [RR; 132-144 mEq/L]). However, potassium and renal function tests were within the normal limits. Three days after admission she was suffering from anasarca, and liver function tests showed low serum albumin 1.4 g/dL (RR; 3.50-5.00 g/dL) and notable coagulopathy with an increased Prothrombin Time and a I.N.R of 2.52 (RR; 1); serum aspartate aminotransferase range was 11-325 UI/L (RR; 12-50 UI/L), alanine aminotransferase range was 1.1-55 UI/L (RR; 10-40 UI/L), γ-glutamyl transpeptidase 24.3 UI/L (RR; 10-40 UI/L), total bilirubin range 2.59-8.75 mg/dL (RR; 0.20-1.00 mg/dL), alkaline phosphatase 38.54 UI/L (RR; 50-136 UI/L), and C-reactive protein 47.72 mg/L (RR;< 5 mg/L). Serum immunoglobulin levels were: IgG 169 mg/dL (RR; 100-360 mg/dL), IgM 14 mg/dL (RR; 26-122 mg/dL), IgA 3 mg/dL (RR; 7-37 mg/dL), and IgE 0.71 (UI/mL, <1.5). Serologic testing for syphilis was negative. A second abdominal ultrasound revealed retroperitoneal lymphadenopathy and ascites, and the kidneys were normal. The clinical course following admission was of rapid deterioration with worsening hepatomegaly, hyperbilirubinemia, abdominal distention, abdominal circumference 43.5 cm [fig_ref] Figure 1: A -Gross examination of the corpse showing marked abdominal distention [/fig_ref] and ascites, radiological thoracoabdominal images showed bilateral diaphragm elevation [fig_ref] Figure 1: A -Gross examination of the corpse showing marked abdominal distention [/fig_ref] and intestinal distention. On the fourth day a limited bone marrow aspirate showed no abnormal infiltrate or hemophagocytosis, and an excisional skin biopsy was taken. The patient died on the fifth day with respiratory distress syndrome (respiratory rate 70 breaths per minute), without evidence of hemorrhage. ## Pathologic findings Due to the working diagnosis of congenital leukemia, a skin excisional biopsy was received and was evaluated immediately by fine needle aspiration (FNA) (caliber 26), which disclosed large histiocytic-like cells [fig_ref] Figure 2: Cytological example obtained by FNA of the skin biopsy [/fig_ref] ; therefore, the diagnosis of histiocytosis was suggested. The surgical specimen was studied on formalin-fixed and paraffin-embedded tissue sections. Light microscopic study at low magnification revealed a dense cellular nodule poorly demarcated involving the entire dermis. At higher magnification, the predominant cells appeared to be histiocytes, with occasional eosinophils. There were few Touton giant cells [fig_ref] Figure 3: Photomicrographs of the skin biopsy showing dermal expansion for infiltration of histiocytes... [/fig_ref] , but no cells with foamy cytoplasm, nuclear atypia, or mitotic figures. ## T h e d i a g n o s i s o f j x g w a s m a d e . Immunohistochemical staining showed that all histiocytes were positive for CD68, CD163, and Factor XIIIa, and negative for S-100, CD1a, and langerin [fig_ref] Figure 4: Photomicrographs of the skin biopsy [/fig_ref] (CD68/KP-1:700/Biocare, CD163/1:50/BioSb, Factor XIIIa/1:200/Biocare, S-100/1:3800/Dako, CD1a/1:500/Dako, and langerin/1:40/BioSb, respectively). Autopsy permission was restricted to the thoracic and abdominal organs. The infant had generalized edema, an abdominal circumference of 43.5 cm, ascites, and mild jaundice. The postmortem examination revealed intense small bowel distention; the liver weighed 500 g (RR; 121 ±39.2 g); and was extensively infiltrated by irregular micronodular tumor [fig_ref] Figure 5: Gross view of the liver showing the parenchyma diffusely infiltrated by the... [/fig_ref]. At the cut surface, the liver gave off a fetid odor. The histology showed that tumor cells consisted of no plump histiocytes and spindle cell forms, and a few Touton giant cells were present. The tumor obliterated many portal and hepatic veins. In the liver, the tumor was preferentially located within the portal triads, but the biliary epithelium was spared [fig_ref] Figure 6: Photomicrographs of the liver [/fig_ref]. Macroscopically the spleen was apparently free of tumor, but microscopically there were abundant plump histiocytic cells, positive for CD163, which obliterated the sinusoids [fig_ref] Figure 7: Photomicrographs of the spleen [/fig_ref]. Nodular tumor lesions were also present in the thoracic lymph nodes, pleura [fig_ref] Figure 8: Gross back view of the cardiopulmonary block showing yellowish pleural nodes in... [/fig_ref] , mesenteric lymph nodes, perirectal fat [fig_ref] Figure 9: A -Gross view of the mesenteric and perirectal lymph nodes [/fig_ref] , pancreas [fig_ref] Figure 1: A -Gross examination of the corpse showing marked abdominal distention [/fig_ref] , periadrenal, and both kidneys [fig_ref] Figure 1: A -Gross examination of the corpse showing marked abdominal distention [/fig_ref]. Multiple bacterial colonies were identified in the hepatic, splenic, and pulmonary vascular spaces [fig_ref] Figure 1: A -Gross examination of the corpse showing marked abdominal distention [/fig_ref] , however, blood cultures were not performed. Other relevant findings were pulmonary atelectasis and mild pulmonary hypoplasia [fig_ref] Figure 1: A -Gross examination of the corpse showing marked abdominal distention [/fig_ref]. Death was attributed to sepsis and respiratory distress syndrome secondary to pulmonary hypoplasia worsened by severe abdominal distention. # Discussion The aid of recent advances in immunohistochemistry enabled better classification of dendritic and histiocytic cell neoplasms and improved the knowledge of tumor biology and histogenesis, which may be helpful in the management of these rare diseases. Dermal and interstitial dendrocytes are responsible for JXG. 2,3 JXG and Langerhans cell histiocytosis (LCH), together in the category of "dendritic cell-related proliferations", currently are separated from the macrophage-related proliferations. The reported cases with the coexistence of JXG and LCH suggest their common origin. [bib_ref] A child with coexistent juvenile xanthogranuloma and Langerhans cell histiocytosis, Yu [/bib_ref] [bib_ref] Langerhans cell histiocytosis preceding the development of juvenile xanthogranuloma: A case and..., Bains [/bib_ref] Patients with JXG and neurofibromatosis types 1 and 2, as well as a triad with juvenile chronic myelogenous leukemia, has been reported.In contrast to other Xanthomatous diseases, JXG is a normolipemic disorder. [bib_ref] Congenital disseminated juvenile xanthogranuloma with unusual skin presentation and renal involvement, Kolivras [/bib_ref] [bib_ref] Eruptive xanthomas, Zaremba [/bib_ref] The Kiel Pediatric Tumor Registry reported 129 cases of JXG over 36 year (0.52% among 24,600 children), showing the very low frequency of the disease. 8 JXG affects mainly children younger than 2 years of age. [bib_ref] Juvenile xanthogranuloma in childhood and adolescence: a clinicopathologic study of 129 patients..., Janssen [/bib_ref] [bib_ref] Juvenile xanthogranuloma: clinicopathologic analysis and immunohistochemical study of 57 patients, Sonoda [/bib_ref] In the analysis of Janssen and Harms, [bib_ref] Juvenile xanthogranuloma in childhood and adolescence: a clinicopathologic study of 129 patients..., Janssen [/bib_ref] there were 71.3% of patients younger than 12 months; in contrast, Dehner [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] reported a frequency of 45% within the first year. According to various authors, the male to female ratio goes from "1.1:1" to .7:1". [bib_ref] Juvenile xanthogranuloma in childhood and adolescence: a clinicopathologic study of 129 patients..., Janssen [/bib_ref] However, when considering only newborns, the ratio of male to female is 1:2.4 [fig_ref] Table 1: Reported cases of infantile systemic juvenile xanthogranuloma, detected at birth, with liver... [/fig_ref]. Clinically there is a broad spectrum of differential diagnoses, including reactive processes, or benign or malignant tumors. The literature is replete with single-case reports of cutaneous JXG-the most common form of non-LCH of childhood-but JXG may occur at any site of the body, and only 4-5% of children with JXG have the infantile systemic variant of the disease. [bib_ref] Juvenile xanthogranuloma in childhood and adolescence: a clinicopathologic study of 129 patients..., Janssen [/bib_ref] [bib_ref] Juvenile xanthogranuloma: forms of systemic disease and their clinical implications, Freyer [/bib_ref] [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] [bib_ref] Fatal congenital systemic juvenile xanthogranuloma with liver failure, Hu [/bib_ref] [bib_ref] Congenital systemic juvenile xanthogranuloma with placental lesion, Takeuchi [/bib_ref] [bib_ref] Prophylactic recombinant factor VIIa administration to an infant with congenital systemic juvenile..., Santiago [/bib_ref] [bib_ref] Neonatal systemic juvenile xanthogranuloma with ominous presentations and successful treatment, Fan [/bib_ref] [bib_ref] Systemic form of juvenile xanthogranuloma: Report of a case with liver and..., Chantranuwat [/bib_ref] A PubMed search revealed very few congenital and lethal ISJXG cases similar to ours [fig_ref] Table 1: Reported cases of infantile systemic juvenile xanthogranuloma, detected at birth, with liver... [/fig_ref] , with none having more than five affected sites; however, in our case, there were eight affected sites. In ISJXG, the most important morphologic differential diagnosis is with LCH, the most common form of dendritic cell proliferations. [bib_ref] Other histiocytic and dendritic cell neoplasms, Chang [/bib_ref] [bib_ref] Dendritic cell and histiocytic neoplasms: biology, diagnosis, and treatment, Dalia [/bib_ref] [bib_ref] Clinicopathologic characteristics and outcomes of histiocytic and dendritic cell neoplasms: the Moffitt..., Dalia [/bib_ref] In ISJXG, the skin is involved in only 50% of cases, and the liver is involved in most of these cases, with predominantly portal infiltrate spilling over into the adjacent lobule but sparing the biliary tree, which contrasts with LCH. 7 As Fan and Sun 30 reported, when JXG has liver involvement, it is a relatively benign disease in its worst form. The location and character of hepatic lesions are important clues to the differential diagnosis, as reported by Favara. [bib_ref] Histopathology of the liver in histiocytosis syndromes, Favara [/bib_ref] Other involved sites include: soft tissue, upper aerodigestive tract, CNS, lymph node, bone marrow, kidneys, and lung; other more unusual sites affected are spleen, myocardium, retroperitoneum, pancreas, and adrenal glands. [bib_ref] Update on juvenile xanthogranuloma: unusual cutaneous and systemic variants, Chang [/bib_ref] [bib_ref] Liver involvement in the histiocytic disorders of childhood, Jaffe [/bib_ref] From the 174 JXG cases reviewed by Denher, [bib_ref] Juvenile xanthogranulomas in the first two decades of life: a clinicopathologic study..., Dehner [/bib_ref] 8 had systemic disease, 2 were diagnosed at birth with hepatosplenomegaly and died of acute hepatic failure, but 1 survived after systemic chemotherapy. From 1996 to 2011 there were at last 31 informed cases of the clinical variant ISJXG; in 17 cases the condition was present at birth, 6 of these cases were fatal (35.2%) [fig_ref] Table 1: Reported cases of infantile systemic juvenile xanthogranuloma, detected at birth, with liver... [/fig_ref]. The reported ISJXG fatal cases of the neonates had a similar clinical presentation to our patient. This supports the concept that ISJXG, complicated by the hepatic infiltration in the neonatal period, is a life-threatening disease caused by hepatic failure, or mainly because severe hepatosplenomegaly induced respiratory failure, as happened in our case. The severe hepatosplenomegaly in our case could have been present since late gestation and compressed the thoracic cavity resulting in mild pulmonary hypoplasia by upward compression the diaphragm-a finding not previously reported in ISJXG. This patient with JXG, which was apparently limited to the skin, must have had a complete physical examination to detect the systemic form, with laboratory and imaging studies if other abnormalities were suspected. Diagnostic studies may include computed tomography or magnetic resonance imaging (MRI), ultrasonography of the abdomen, a radionuclide bone scan, and a detailed ophthalmologic examination. In the instance of neonatal ISJXG where a quick diagnosis is required, FNA cytology made from the surgical biopsies is a useful tool for the initial differential diagnostic procedure and management. [bib_ref] Deep-seated congenital juvenile xanthogranuloma: report of a case with emphasis on cytologic..., Barroca [/bib_ref] [bib_ref] Cytologic features of deep juvenile xanthogranuloma, Grenko [/bib_ref] [bib_ref] Neonatal juvenile xanthogranuloma: report of a case with fine needle aspiration cytologic..., Gutiérrez Martín [/bib_ref] In our case, with the working diagnosis of congenital leukemia, the cytology of the skin biopsy allowed the proposed diagnosis of histiocytosis within a few minutes. Clinical presentations include anemia and thrombocytopenia; however, there is little reliable information on their natural history or the treatment of choice. The vast majority of congenital ISJXG cases, even those with visceral involvement, experience disease regression without specific treatment; 4 however, severe morbidity has been reported in some cases, which need supportive interventions and chemotherapy. [bib_ref] Juvenile xanthogranuloma: forms of systemic disease and their clinical implications, Freyer [/bib_ref] [bib_ref] Fatal congenital systemic juvenile xanthogranuloma with liver failure, Hu [/bib_ref] [bib_ref] Juvenile xanthogranuloma: clinicopathologic analysis and immunohistochemical study of 57 patients, Sonoda [/bib_ref] Treatment protocols recommended for patients with symptoms who have unresectable lesions are those used for LCH. The LCH-III protocol-the most common treatment strategy-was first suggested by Nakatani et al. [bib_ref] Successful treatment of congenital systemic juvenile xanthogranuloma with Langerhans cell histiocytosis-based chemotherapy, Nakatani [/bib_ref] Although most patients with the multisystemic disease are infants and particularly susceptible to the adverse effects of chemotherapy, a prudent approach should emphasize the supportive care, reserving chemotherapy or radiation when this treatment is perceived as necessary. Immunophenotypically, cells express CD14, CD68, CD163, Factor XIIIa, Stabilin-1, and fascin; S100 is a variable positive in less than 20% of the cases; nevertheless, none of these markers are specific for ISJXG,and CD1a and langerin are negative. In our case, CD163 and Factor XIIIa were positive, while S100 and CD1a were negative. No consistent cytogenetic or molecular genetic alterations have been identified. [bib_ref] Fatal congenital systemic juvenile xanthogranuloma with liver failure, Hu [/bib_ref] [bib_ref] Systemic juvenile xanthogranuloma with fatal outcome, Azorín [/bib_ref] CONCLUSIONS Infantile systemic forms of JXG have different presentation, response to the treatment and outcome. Incidence rates are about 0.1 case/1,000.000 of children younger than 15 years old. Although this report has the inherent limitation of a case report, it presents the importance of the autopsy examination, which can still influence medical practice and show several problems that were clinically undetected. In this case, the disseminated neoplasms could have been diagnosed earlier, because, in neonatal patients in whom JXG is considered, systemic forms could be underestimated, as in this case. For new patients with cutaneous JXG who have other symptoms, clinicians must be alert to refer the patient to a tertiary care center where tests, including (at the very least) MRI of the brain and ultrasonography of the abdomen, can be undertaken to enable accurate diagnosis. Adjuvant chemotherapy could have changed the course of the disease in our case. Although JXG is the most common benign type of histiocytosis in infants, the ISJXG variant is a very rare and distinctive clinicopathologic entity, generating a challenging group of patients. More than 70% of the patients are younger than 1 year. ISJXG must be distinguished principally from LCH. The clinical findings are attributable to sites of disease and can be severe. No published case has eight affected sites of the body, as in our case. The most common sites of disease beyond subcutaneous soft tissue are liver, spleen (or both), lung, and CNS. Exceptionally, liver, spleen, or lung involvement cause serious consequences, and the gold standard for diagnosis is the biopsy. The respiratory distress and bacteremia, as in our case, may be the cause of death in most cases. [fig] Figure 1: A -Gross examination of the corpse showing marked abdominal distention (abdominal circumference 43.5 cm). Note the skin nodules (arrows) on the upper left extremity and lower right extremity, and genital edema; B -Plain thoraco-abdominal radiograph demonstrating the enlarged liver and diaphragm elevation. [/fig] [fig] Figure 2: Cytological example obtained by FNA of the skin biopsy. Disclosed monotonous histiocytic type cells. Cytologic features allowed us to suggest the diagnosis of histiocytosis. FNA = fine needle aspiration. (H&E stain), A (100 X), B (400 X). [/fig] [fig] Figure 3: Photomicrographs of the skin biopsy showing dermal expansion for infiltration of histiocytes and occasional Touton giant and eosinophil cells (H&E stain) A (40X), B (100X), C (400 X), and D (400_X). [/fig] [fig] Figure 4: Photomicrographs of the skin biopsy. Immunohistochemistry was positive for CD68 (A) and Factor XIIIa (C), and negative for CD1a (B) and S-100 (D). [/fig] [fig] Figure 5: Gross view of the liver showing the parenchyma diffusely infiltrated by the tumor. [/fig] [fig] Figure 6: Photomicrographs of the liver. A, and C show the portal tract with diffuse infiltration by histiocytes and in B a central vein. The bile ducts are not affected, which is clearly seen with CD163. A (H&E, 100X), B (400X) and C (100X). [/fig] [fig] Figure 7: Photomicrographs of the spleen. A and B -High magnification of multisystemic juvenile xanthogranuloma with diffuse infiltration by plump histiocytes, which is strongly positive for CD163. A (H&E, 400X) and B (100X). [/fig] [fig] Figure 8: Gross back view of the cardiopulmonary block showing yellowish pleural nodes in both lungs. [/fig] [fig] Figure 9: A -Gross view of the mesenteric and perirectal lymph nodes (C). The lymph nodes are infiltrated by foamy macrophages and Touton giant cells (B and D respectively) (H&E, B(40X) and D(400X). [/fig] [fig] *: Previously reported by Freyer et al. J Pediatr 1996;129:227-37; 6-MP = 6-mercaptopurine; ADF = alive disease-free; AWD = alive with disease; BM = bone marrow; CNS = central nervous system; CSA = cyclosporine; DOD = dead of disease; DX = dexamethasone; F = female; Ig = immunoglobulins; LCH = Langerhans cell histiocytosis; M = male; MPN = metilprednisolone; MTX = methrotexate; PDN = prednisone; RT = radiation therapy; VA = Vinca alkaloids; VBL = vinblastine; VP16 = etopsoside. [/fig] [table] Table 1: Reported cases of infantile systemic juvenile xanthogranuloma, detected at birth, with liver affection (n=17), searched in PubMed. Six cases (35.2%) were fatal [/table]

FAIR data representation in times of eScience: a comparison of instance-based and class-based semantic representations of empirical data using phenotype descriptions as example Background: The size, velocity, and heterogeneity of Big Data outclasses conventional data management tools and requires data and metadata to be fully machine-actionable (i.e., eScience-compliant) and thus findable, accessible, interoperable, and reusable (FAIR). This can be achieved by using ontologies and through representing them as semantic graphs. Here, we discuss two different semantic graph approaches of representing empirical data and metadata in a knowledge graph, with phenotype descriptions as an example. Almost all phenotype descriptions are still being published as unstructured natural language texts, with far-reaching consequences for their FAIRness, substantially impeding their overall usability within the life sciences. However, with an increasing amount of anatomy ontologies becoming available and semantic applications emerging, a solution to this problem becomes available. Researchers are starting to document and communicate phenotype descriptions through the Web in the form of highly formalized and structured semantic graphs that use ontology terms and Uniform Resource Identifiers (URIs) to circumvent the problems connected with unstructured texts.Results: Using phenotype descriptions as an example, we compare and evaluate two basic representations of empirical data and their accompanying metadata in the form of semantic graphs: the class-based TBox semantic graph approach called Semantic Phenotype and the instance-based ABox semantic graph approach called Phenotype Knowledge Graph. Their main difference is that only the ABox approach allows for identifying every individual part and property mentioned in the description in a knowledge graph. This technical difference results in substantial practical consequences that significantly affect the overall usability of empirical data. The consequences affect findability, accessibility, and explorability of empirical data as well as their comparability, expandability, universal usability and reusability, and overall machine-actionability. Moreover, TBox semantic graphs often require querying under entailment regimes, which is computationally more complex.Conclusions: We conclude that, from a conceptual point of view, the advantages of the instance-based ABox semantic graph approach outweigh its shortcomings and outweigh the advantages of the class-based TBox semantic graph approach. Therefore, we recommend the instance-based ABox approach as a FAIR approach for documenting and communicating empirical data and metadata in a knowledge graph. as classes and class axioms 5 that are defined through universal statements [bib_ref] A study of big data and its challenges, Idrees [/bib_ref] [bib_ref] Strengths and limitations of formal ontologies in the biomedical domain, Schulz [/bib_ref] [bib_ref] Formal ontologies in biomedical knowledge representation, Schulz [/bib_ref] , ontologies consist of TBox expressions and not ABox expressions and thus do not contain statements about individual entities. Ontologies in this sense, therefore, do not contain actual empirical data. But one can employ ontology terms in ABox expressions, for instance for stating that a given individual entity is of a particular kind and that it therefore represents an instance of the respective ontology class. Each ontology class, individual entity, and property possesses its own unique and persistent Uniform Resource Identifier 7 (URI), through which it can be identified and individually referenced in various contexts. By providing URIs and machine-readable definitions for their classes, ontologies can be used to substantially increase semantic transparency and machine-actionability for all kinds of information, including empirical data. The URIs of ontology classes are often used for semantically enriching documents for data mining purposes of historical literature and for annotating database contents to improve integration and interoperability of data and thus computability of contemporary empirical data. Using Semantic Web technologies, ontologies can be employed to express, document, and represent empirical data as structured, interlinked, and semantically rich semantic graphs that substantially improve the findability, accessibility, interoperability, and reusability of data, thus making data compliant with the FAIR Guiding Principles [bib_ref] The FAIR guiding principles for scientific data management and stewardship, Wilkinson [/bib_ref]. This is becoming increasingly important in the age of Big Data and Linked Open Data and allows data and metadata to be used in eScience [bib_ref] eScience and the need for data standards in the life sciences: in..., Vogt [/bib_ref] [bib_ref] On the importance of standardisation in life sciences, Brazma [/bib_ref] [bib_ref] Minimum information about a microarray experiment (MIAME)-toward standards for microarray data, Brazma [/bib_ref] [bib_ref] From XML to RDF: how semantic web technologies will change the design..., Wang [/bib_ref] [bib_ref] A strategy capitalizing on synergies: the reporting structure for biological investigation (RSBI)..., Sansone [/bib_ref] [bib_ref] The need for data standards in zoomorphology, Vogt [/bib_ref]. Ontologies are thus cornerstones of the Semantic Web and provide solutions to various problems of information and knowledge management, including word-sense disambiguation, standardization, and measuring semantic similarity, providing an efficient framework for question answering, knowledge representation, natural language processing, and semantic searches [bib_ref] Lineage path integration for phylogenetic resources, Herbert [/bib_ref] [bib_ref] Semantic similarity in biomedical ontologies, Pesquita [/bib_ref] [bib_ref] Semantic annotation of morphological descriptions: an overall strategy, Cui [/bib_ref] [bib_ref] CharaParser for fine-grained semantic annotation of organism morphological descriptions, Cui [/bib_ref] [bib_ref] Applications of natural language processing in biodiversity science, Thessen [/bib_ref] [bib_ref] Similarity-based search of model organism, disease and drug effect phenotypes, Hoehndorf [/bib_ref] [bib_ref] Measuring phenotype-phenotype similarity through the interactome, Peng [/bib_ref] [bib_ref] InfAcrOnt: calculating cross-ontology term similarities using information flow by a random walk, Cheng [/bib_ref] [bib_ref] Annotation of phenotypes using ontologies: a gold standard for the training and..., Dahdul [/bib_ref]. ## Real particulars, real universals, and their textual representations Documenting empirical data in the form of semantic graphs attempts to represent a particular portion of reality and is, as such, a semiotic process. When reflecting on the way we do research and especially when comparing different approaches of representing empirical reality, it is good to distinguish basic categories of entities that are involved in this semiotic process [bib_ref] eScience and the need for data standards in the life sciences: in..., Vogt [/bib_ref] [bib_ref] Assessing similarity: on homology, characters and the need for a semantic approach..., Vogt [/bib_ref] [bib_ref] Towards a Reference Terminology for Ontology Research and Development in the Biomedical..., Smith [/bib_ref] [bib_ref] Signs and terminology: science caught between language and perception, Vogt [/bib_ref]. Empirical data attempt to represent real entities and their relations. Real entities are material objects, processes, qualities, and states that exist in reality, independent of any human mind. Any real entity is either a particular (i.e., instance, individual, token) or a universal (i.e., kind, type). Particulars-e.g. the planet Earth or you, the reader, and I-are singly located entities that are bound to a specific location in space and time, whereas universals-e.g. cell or multicellular organismare multiply located entities that exist in their corresponding particulars [bib_ref] On substances, accidents and universals -in defence of a constituent ontology, Smith [/bib_ref] [bib_ref] The logic of biological classification and the foundations of biomedical ontology, Smith [/bib_ref]. A universal is thus anything that is instantiated by particulars and a particular anything that instantiates a universal [bib_ref] Beyond concepts: ontology as reality representation, Smith [/bib_ref]. In this sense, I am an instance of multicellular organism. Real entities do not exist inside of our minds but outside in the real world. When we think of a real entity, we generate a cognitive representation referring to it in the form of thoughts, perceptions, concepts, ideas, and beliefs. When we communicate information about a real entity with somebody else, we want that person to share a maximally similar cognitive representation about the entity. In doing so, we often use language and thus terms and statements for describing our cognitive representations. Any term and statement referring to a real entity is a textual representation of a real entity and must be distinguished not only from the real entity it refers to but also from the cognitive representation it should induce. Based on the distinction of the two basic categories of real entities, i.e., particulars and universals, we can distinguish between textual representations of particulars in the form of proper names and assertional statements and textual representations of universals in the form of kind terms (also called general terms) and universal statements. Proper names refer to particulars and usually have no textual definitions but only assertional statements associated with them. Assertional statements are statements that claim to be only true for a specific particular. If assertional statements are grounded in empirical knowledge that is based on observation and experimentation, we refer to them as empirical data. Empirical data can be formulated in OWL and documented in the form of instance-based ABox semantic graphs, in which particular real entities can be referred to through assigning them their own URIs, and their 5 Ontology classes are described using axioms, i.e., propositions that define the class in a logical form, specifying the necessary and sufficient conditions for an individual entity to be an instance of that class. [bib_ref] A study of big data and its challenges, Idrees [/bib_ref] A universal statement is a proposition that states that all entities of a specific kind have a particular property. A proposition stating that a given individual entity is an instance of a specific kind of entity that has specific properties, on the other hand, represents a particular empirical observation. [bib_ref] The FAIR guiding principles for scientific data management and stewardship, Wilkinson [/bib_ref] A URI unambiguously identifies a particular Web resource, i.e., any identifiable digital, physical, or abstract thing. Uniformity of URIs is guaranteed through a predefined set of syntax rules. The most common form of URI is the Uniform Resource Locator (URL). class affiliation can be specified by referencing the URI of the respective ontology class. A kind term, on the other hand, is usually associated with a concept in the form of a class that defines the meaning of the term by means of universal statements. A universal statement represents commonly accepted domain knowledge and claims to be true for all instances of the kind the statement is referring to. Scientific theories, but also definitions of ontology classes using axioms [bib_ref] The role of ontologies in biological and biomedical research: a functional perspective, Hoehndorf [/bib_ref] , are examples of universal statements. Ontologies contain universal statements that can be formulated in OWL and documented in the form of classbased TBox semantic graphs. These two types of textual representations can for instance be applied to describing anatomical phenotypes. The resulting descriptions attempt to represent the organization of real anatomical entities (cf. anatomical entity 8 of Uber Anatomy Ontology; id UBERON:0001062) in the form of textual representations. Each description consists of at least one descriptive statement. We here understand a descriptive statement as the smallest semantically meaningful unit of empirical information. Descriptive statements can be differentiated based on their semantic content into assertional and universal statements [bib_ref] Strengths and limitations of formal ontologies in the biomedical domain, Schulz [/bib_ref] [bib_ref] Formal ontologies in biomedical knowledge representation, Schulz [/bib_ref] , e.g. the description of the essential properties 9 of a compound eye as a set of universal statements defining the class compound eye or the description of an individual compound eye possessing a particular set of properties documented in a set of assertional statements. For reasons of efficiency and simplicity, descriptions of particulars usually always involve references to ontology classes. Stating that a given particular entity is an instance of the class compound eye, for example, implies that all defining properties of the class 'compound eye' also necessarily apply to this particular entity. To which degree information is provided through class affiliations thereby depends on several factors, including the anatomical variability of the Operational Descriptive Unit (ODU), [bib_ref] SOCCOMAS: a FAIR web content management system that uses knowledge graphs and..., Vogt [/bib_ref] the frame of reference of the description and which relevant ontology terms are available. However, since an instance of a class necessarily has all the classdefining properties, the reference to ontology classes within a description logically and semantically represents an implicit short form for what can be explicitly expressed in an instance-based ABox semantic graph. Regardless, empirical data necessarily and always have to include some ABox expression, even if this may only be a statement about some individual entity instantiating some ontology class. Here, we discuss and evaluate the conceptual differences between two approaches of semantically representing empirical data using semantic graphs, i.e., an instance-based approach that represents data in the form of ABox expressions and a class-based approach that represents them in the form of TBox expressions. We use phenotype descriptions as an example for this comparison. Phenotypes, canonical anatomy, instance anatomy, and the use of ontologies for documenting phenotype descriptions The Phenotype of an organism refers to its observable constituents, properties, and relations that can be considered to result from the interaction of the organism's genotype with itself and its environment. Anatomy is the part of the phenotype that refers to the physical and structural properties of the organism. Anatomical data are the primary source of evidence for defining most species, for understanding their phylogeny, for recognizing, defining, and diagnosing pathological conditions in plants, animals, and other organisms, and they provide valuable insights into the development, function, evolution, and interaction of phenotypes with their environments [bib_ref] Finding our way through phenotypes, Deans [/bib_ref] [bib_ref] Phenotypes in insect biodiversity research, Mikó [/bib_ref]. In anatomy, we distinguish canonical anatomy and instantiated anatomy. Canonical anatomy is "a field of anatomy (science) that comprises the synthesis of generalizations based on anatomical observations that describe idealized anatomy (structure)", whereas instance anatomy is "the field of anatomy (science) which comprises anatomical data pertaining to instances (i.e., individuals) of organisms and their parts" [bib_ref] A reference ontology for biomedical informatics: the foundational model of anatomy, Rosse [/bib_ref] p. 480; see also, e.g., [bib_ref] Motivation and organizational principles for anatomical knowledge representation: the digital anatomist symbolic..., Rosse [/bib_ref] [bib_ref] The foundational model of anatomy ontology, Rosse [/bib_ref]. While instance anatomy aims at representing the actual anatomical organization of a particular organism or a particular anatomical entity as it can be observed, resulting in what could be called 'factual' descriptions [bib_ref] eScience and the need for data standards in the life sciences: in..., Vogt [/bib_ref] [bib_ref] Signs and terminology: science caught between language and perception, Vogt [/bib_ref] , canonical anatomy aims at representing the typical anatomical organization of the members of a certain taxon or typical exemplar instances of a specific kind of anatomical entity. Canonical anatomy is applied in contexts in which deviation from a defined 'normal' condition is important, for instance, in medical contexts or when studying mutants against a canonical wild-type [bib_ref] A logical foundation for representation of clinical data, Campbell [/bib_ref] [bib_ref] Toward a medical-concept representation language, Evans [/bib_ref] [bib_ref] Diurnal changes of polysome loading track sucrose content in the rosette of..., Pal [/bib_ref] [bib_ref] Analysis of recombinant H7N9 wild-type and mutant viruses in pigs shows that..., Liu [/bib_ref] [bib_ref] Comparative metabolic flux analysis of an Ashbya gossypii wild type strain and..., Jeong [/bib_ref]. Information typically belonging to canonical anatomy is commonly accepted domain knowledge in the form of universal statements about kinds of anatomical entities that can be found in ontologies and can be represented as class-based TBox semantic graphs (see also invariants in, whereas information typically belonging toThroughout the paper, we use regular underlined for representing ontology classes with their labels and italic underlined for representing ontology properties with their labels. [bib_ref] eScience and the need for data standards in the life sciences: in..., Vogt [/bib_ref] Essential properties of a specific kind of entity refer to the properties that all instances of the kind must have. [bib_ref] SOCCOMAS: a FAIR web content management system that uses knowledge graphs and..., Vogt [/bib_ref] Analogue to the Operational Taxonomic Unit (OTU), which refers to some taxonomic entity, the ODU refers to the object being described. It is the referent of the description and, in the case of anatomical phenotype descriptions, refers to some anatomical entity. instance anatomy is empirical anatomical data in the form of assertional statements about particular anatomical entities that can be found in phenotype descriptions of individual specimens and can be represented as instance-based ABox semantic graphs. Unfortunately, despite their importance to life sciences and beyond, anatomical data are usually still published as anatomical descriptions using natural language and thus in the form of unstructured texts. The descriptions are not machine-actionable and often hidden behind pay-walls. This substantially impedes the findability and accessibility of anatomical data. Moreover, due to the immanent semantic ambiguity of anatomical terminology, researchers not familiar with the described taxon and its associated anatomical literature will have substantial problems comprehending and interpreting anatomical data [bib_ref] The linguistic problem of morphology: structure versus homology and the standardization of..., Vogt [/bib_ref]. The meaning of terms is often taxon-, author-, and time-dependent. And while some terms refer to a set of common spatio-structural properties, others refer to a common function, a common developmental pathway, or a presumed common evolutionary origin, or some mixture of these. The same applies to phenotype descriptions in general. The semantic ambiguity of phenotype descriptions that are based on natural language substantially limits the interoperability and reusability of phenotype data, with the consequence that phenotype data usually do not comply with the FAIR guiding principles. It has been demonstrated that phenotype descriptions can be represented using ontology terms with RDF's triple syntax of Subject, Predicate, and Object and stored as semantic graphs [bib_ref] The linguistic problem of morphology: structure versus homology and the standardization of..., Vogt [/bib_ref] [bib_ref] Representing Phenotypes in OWL, Mungall [/bib_ref] [bib_ref] Learning from Linnaeus: towards developing the foundation for a general structure concept..., Vogt [/bib_ref] [bib_ref] Phenex: ontological annotation of phenotypic diversity, Balhoff [/bib_ref] [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref] [bib_ref] Evolutionary characters, phenotypes and ontologies: curating data from the systematic biology literature, Dahdul [/bib_ref] [bib_ref] The human phenotype ontology project: linking molecular biology and disease through phenotype..., Köhler [/bib_ref] [bib_ref] The Zebrafish Information Network: the zebrafish model organism database provides expanded support..., Sprague [/bib_ref] [bib_ref] Phenotype ontologies: the bridge between genomics and evolution, Mabee [/bib_ref]. Two alternative basic approaches have been employed for representing the anatomical organization of a given specimen using ontology terms: a class-based TBox and an instance-based ABox approach. The applicability of these two approaches is not limited to phenotypic data but can be used for representing any type of empirical data. The two approaches differ mainly in technical details that have substantial practical consequences in terms of their respective applicability and can also be aligned to underlying conceptual differences resulting from different research contexts. In the class-based TBox approach, a specific anatomical phenotype is described in reference to a specific ontology class, which in turn is defined according to the set of properties that are characteristic to the respective phenotype . The definition of the class takes the form of an Entity-Quality (EQ) expression and provides the description for that particular phenotype in the form of a set of TBox expressions. Respective descriptions have been called Semantic Phenotypes, in which the ODU is specified as instantiating a specific phenotype ontology class [bib_ref] Representing Phenotypes in OWL, Mungall [/bib_ref] [bib_ref] Phenex: ontological annotation of phenotypic diversity, Balhoff [/bib_ref] [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref] [bib_ref] Evolutionary characters, phenotypes and ontologies: curating data from the systematic biology literature, Dahdul [/bib_ref]. In the instance-based ABox approach, the anatomical phenotype is not described within the definition of a single ontology class, but instead in the form of a detailed semantic graph, built from ABox expressions that consist of several instance resources (i.e., URIs), each of which refers to a particular part or property/quality of the ODU . The resources themselves thus represent instances and not classes, but they instantiate ontology classes. We term the resulting descriptions Phenotype Knowledge Graphs, and they follow a more modular framework that makes use of anatomical entity terms and property/quality terms from existing ontologies [bib_ref] Assessing similarity: on homology, characters and the need for a semantic approach..., Vogt [/bib_ref] [bib_ref] The linguistic problem of morphology: structure versus homology and the standardization of..., Vogt [/bib_ref] [bib_ref] Learning from Linnaeus: towards developing the foundation for a general structure concept..., Vogt [/bib_ref] [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref] [bib_ref] The logical basis for coding ontologically dependent characters, Vogt [/bib_ref] [bib_ref] Towards a semantic approach to numerical tree inference in phylogenetics, Vogt [/bib_ref]. In the following, we start by specifying the requirements that empirical data must satisfy in the age of eScience and Big Data, with phenotype data as an example. Based on these distinctions and requirements, we introduce and compare the class-based and the instancebased approach for documenting empirical data in the form of semantic graphs. We provide some historical background on how the class-based approach evolved to explain why certain conceptual choices have been made. We discuss the contexts in which the two approaches can be applied and discuss and evaluate the technical differences between them and their practical consequences within the field of anatomy. We think that with their considerable complexity and heterogeneity, covering quantitative (measurements) as well as qualitative (form, shape) information, including contextual information in the form of descriptions of a specimen relative to some other specimen, phenotype descriptions provide a well-suited framework for comparing and evaluating the benefits and shortcomings of instance-based and classbased semantic graphs as two basic approaches of semantically representing empirical data. Finally, we discuss the conceptual suitability of both approaches regarding meeting eScience-compliant standards and the FAIR Guiding Principles. We want to emphasize that we do not intend to compare the overall benefits and problems of representing and using data or knowledge in the form of ABox and TBox expressions in general. We rather focus in our comparison on the context of documenting and managing empirical data and thus the results from observations, measurements, and experimentation. # Methods In the age of eScience and Big Data, empirical data must meet certain technical requirements to be able to take full advantage of the benefits that semantic analytical frameworks offer. They should be easily findable, accessible, and explorable for human readers in respective online data repositories and actionable for machines alike. In case of phenotype data, a researcher should be able to query a phenotype repository using detailed searches, e.g., for descriptions of heads that possess a specific type of antenna and that have a weight larger than 10 mg, restrict this search to a specific taxonomic group and retrieve a list of corresponding descriptions, preferably associated with images supporting the findings. Ideally, we need something comparable to a BLAST search for phenotypes that enables finding descriptions in a repository that are maximally similar to an input description. Not only empirical data themselves should be findable, accessible, and explorable but also all relevant associated metadata, amongst others to be able to evaluate the trustworthiness and credibility of the data. Non-experts should be able to understand and interpret empirical data correctly, just like researchers can do today with DNA sequence data without having to be a molecular biologist by profession. Data representations thus must be semantically transparent in the sense that they make the meaning of terms used in the representation readily available. Data representations should also be comparable, and it should be possible to expand them and complement them with additional and more detailed information. Moreover, because humans make mistakes, we need an effective way in which researchers can correct mistakes in data representations and thereby transparently track what has been changed. It should be possible to integrate different frames of reference (in the case of anatomical data, possible frames of reference would be, i.e., structural, functional, developmental, and evolutionary anatomy) within Phenotype description of an insect head with a flattened shape and with two antennae in the form of a class-based TBox semantic graph, OWL Manchester Syntax, and an instance-based ABox semantic graph. A: The class-based TBox semantic graph description of an insect head with a flattened shape that has two antennae as its parts. It consists of an instance (purple-bordered box) that instantiates the phenotype class that contains the actual description of the phenotype (yellow-bordered box) in the form of a class axiom consisting of anonymous property restrictions and class descriptions (grey-bordered boxes). The class axiom characterizes all instances of the class to consist of exactly one instance of insect head (id UBERON:6000004) that has the quality of a flattened (id PATO:0002254) shape and that has as its parts exactly two instances of antenna (id UBERON:0000972). B: An alternative format representing the class axioms from the 'phenotype class' from (A) expressed in OWL Manchester Syntax(ontology classes shown with their label underlined, ontology properties with their label in italics and underlined, 'and' being used in the sense of intersection of two mathematical sets and 'exactly' as a cardinality specification). C: The instance-based ABox semantic graph description of an insect head with a flattened shape that has two antennae as its parts. It consists of the instance of the class insect head (id UBERON:6000004), the instance of the class flattened (id PATO:0002254), which relates to the head as its quality, and two instances of the class antenna (id UBERON:0000972), which relate to the head as its parts. Labels (in light-grey-bordered boxes) indicate how the different instances should be represented in a human-readable format. For reasons of clarity, resources are not represented with their URIs but with their human-readable labels. Purplebordered box = instance resource; yellow-bordered box with rounded corners = ontology class resource; grey-bordered box with rounded corners = anonymous class; light-grey-bordered box = literal or numerical value; labeled arrow = property resource descriptive empirical data and to fragment a description into smaller parts to reuse only those parts of the description that are of interest for a given context. If we establish a general, domain-specific standard for representing descriptive empirical data, this standard should be universally usable and reusable, i.e., the syntax, format, and other standards associated with descriptive data should not be specifically tailored for a particular analytical framework such as phylogenetics within the domain of anatomy. Researchers should be able to use descriptive data in the scientific context that is relevant to them. Last, but not least, researchers should also be able to generate empirical data in the most time-efficient way. If for instance phenotype descriptions themselves would be completely machine-actionable, we could develop tools and algorithms that facilitate the semiautomatic generation of phenotype descriptions from images, therewith widening one of the most problematic bottlenecks in anatomy: generating anatomical phenotype descriptions and thus anatomical data.If descriptions are machine-actionable, we can also start to parameterize the analysis of phenotype descriptions so that character analysis and character construction no longer represent a black box or remain to be a matter of authority. We can develop algorithms that quantify the degree of similarity between two given descriptions, hence subjecting similarity propositions to constructive criticism and corrections and providing comparative phenotypic methods a mathematical statistical analytical framework [bib_ref] Assessing similarity: on homology, characters and the need for a semantic approach..., Vogt [/bib_ref]. Eventually, the semantic framework could provide the unified theory of character construction that biology is yet lacking [bib_ref] A comparison of two methods of character construction, Wilkinson [/bib_ref]. In the following, we discuss two different approaches to semantically representing phenotype descriptions. We argue that, from a conceptual point of view, the Phenotype Knowledge Graph approach is superior to the Semantic Phenotypes approach because it minimizes the number of TBox expressions necessary for carrying descriptive contents, which brings about the technical advantage of each entity, quality, and relation being referred to in a description having its own URI. As a consequence, these entities, qualities, and properties can be individually identified, which in turn brings about various practical advantages that together better meet the above-mentioned requirements. # Results Semantic phenotypes: phenotype descriptions as classbased TBox representations When researchers started to conduct large-scale mutagenesis screens in model organisms, labs were suddenly able to analyze large collections of mutants. This raised new challenges regarding scale and complexity of the newly generated data and their analysis and interpretation with respect to their relationship to corresponding phenotypes, leading to the use of ontologies for standardizing mutant phenotype descriptions [bib_ref] Phenotype ontologies: the bridge between genomics and evolution, Mabee [/bib_ref] [bib_ref] From genotype to phenotype: linking bioinformatics and medical informatics ontologies, Holloway [/bib_ref] [bib_ref] Connecting evolutionary morphology to genomics using ontologies: a case study from cypriniformes..., Mabee [/bib_ref] [bib_ref] Practical application of ontologies to annotate and analyse large scale raw mouse..., Beck [/bib_ref]. These mutant phenotype descriptions were comparative phenotype descriptions, i.e., phenotype descriptions that are based on comparative observations that characterize the outcomes of an experiment or observed difference against a specific reference state such as the mutant in comparison to the wild-type [bib_ref] The anatomy of phenotype ontologies: principles, properties and applications, Gkoutos [/bib_ref]. The problem with comparative descriptions against some 'normal' condition or state is that they describe instance anatomy in reference to canonical anatomy and in doing so convey information about at least two different entities, i.e., a particular mutant and a 'universal' wild-type. This restricts the usability of the descriptions to the context of comparison against the wild-type, because the direct observation, on which the comparison is based, often cannot be derived anymore. When for instance stating that the described specimen has an "increased length of abdomen", we do not know the actual length of the specimen's abdomen, not even relatively as in "length of abdomen above 2.6 mm". If we want to get any information about that abdomen, we must first consult the description of the wild-type, in order to derive the lower boundary value of the possible length for the described abdomen. With respect to the application of ontologies for standardized mutant-phenotype descriptions, two different class-based approaches were initially followed [bib_ref] Practical application of ontologies to annotate and analyse large scale raw mouse..., Beck [/bib_ref] [bib_ref] Phenobabelomics -mouse phenotype data resources, Hancock [/bib_ref] [bib_ref] Automatically transforming pre-to post-composed phenotypes: EQ-lising HPO and MP, Oellrich [/bib_ref] [bib_ref] The digital revolution in phenotyping, Oellrich [/bib_ref] : 1) The class-based pre-composition approach uses a single dedicated ontology that provides phenotype descriptions in the form of ontology classes for annotating natural language descriptions of phenotypes, with each phenotype having its corresponding ontology class [53] (pre-composed, because the phenotype description is completely covered by the definition of the respective ontology class; reference to the URI of that ontology class is sufficient). The definitions of phenotype classes usually reference a combination of entities and values (e.g., abnormal body weight, id MP:0001259, of the Mammalian Phenotype ontology, MP). A particular phenotype is then described by referencing the URI of a corresponding ontology class [bib_ref] The anatomy of phenotype ontologies: principles, properties and applications, Gkoutos [/bib_ref]. In order to be able to reference a suitable URI, however, the ontology class must be precomposed in advance by the ontology editor of the respective phenotype ontology (see pre-composition [bib_ref] Integrating phenotype ontologies across multiple species, Mungall [/bib_ref]. 2) In 2004, the need for a more systematic and formalized approach was recognized, resulting in the class-based post-composition approach and the development of the Phenotype And Trait Ontology (PATO), a species-neutral ontology of attributes and values [bib_ref] The anatomy of phenotype ontologies: principles, properties and applications, Gkoutos [/bib_ref]. The post-composition approach characterizes and defines phenotypes following a formalized syntax using class expressions from various distinct ontologies and applying the entity-quality (EQ) format [bib_ref] Building mouse phenotype ontologies, Gkoutos [/bib_ref] [bib_ref] Using ontologies to describe mouse phenotypes, Gkoutos [/bib_ref] (post-composed, because the actual phenotype description must be composed from references to several ontology classes and/or values using the EQ format). According to this post-composition approach, one characterizes a phenotype in terms of a bearer entity (E) that is described by a specific quality (Q). The term that defines the bearer entity is provided by a class of some domain ontology, the term defining the specific quality by a class of PATO. The resulting EQ statement completely replaces the natural language description of the phenotype. Originally, the statement took the tripartite structure of Entity + Attribute + Value such as in eye + color + red (EAV or Entity-Attribute approach [bib_ref] Phenobabelomics -mouse phenotype data resources, Hancock [/bib_ref] [bib_ref] Building mouse phenotype ontologies, Gkoutos [/bib_ref] [bib_ref] Using ontologies to describe mouse phenotypes, Gkoutos [/bib_ref]. After the quality terms in PATO have been organized hierarchically, with more specific terms such as red (id PATO:0000322) being subsumed as subclasses under more general terms such as color (id PATO:0000020), the tripartite structure has been adapted to the bipartite structure of Entity + Quality [bib_ref] Representing Phenotypes in OWL, Mungall [/bib_ref] [bib_ref] Phenex: ontological annotation of phenotypic diversity, Balhoff [/bib_ref] [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref] [bib_ref] Phenotype ontologies: the bridge between genomics and evolution, Mabee [/bib_ref] [bib_ref] Connecting evolutionary morphology to genomics using ontologies: a case study from cypriniformes..., Mabee [/bib_ref] [bib_ref] Using ontologies to annotate large-scale mouse phenotype data, Beck [/bib_ref] [bib_ref] Linking human diseases to animal models using ontology-based phenotype annotation, Washington [/bib_ref] [bib_ref] Time to change how we describe biodiversity, Deans [/bib_ref] [bib_ref] The first organ-based free ontology for arthropods (ontology of arthropod circulatory systems..., Wirkner [/bib_ref]. As a result, the natural language phenotype description "eye has red color" translates into the EQ statement eye + red, with, for instance, eye (id MA:0000261) from the Mouse Adult Gross Anatomy Ontology and red (id PATO:0000322) from PATO. An obvious advantage of the post-composition approach is that it limits the number of ontology terms required for describing a given phenotype because annotators have the ability to compose phenotype descriptions on-the-fly using combinations of terms from available ontology classes to form a multiplicity of different EQ statements [bib_ref] Automatically transforming pre-to post-composed phenotypes: EQ-lising HPO and MP, Oellrich [/bib_ref] [bib_ref] Integrating phenotype ontologies across multiple species, Mungall [/bib_ref]. The bipartite structure of the EQ statements also lends itself for being stored in a table of a relational database, with E and Q each providing a value in the form of a URI for a corresponding cell in the table. However, phenotype annotations in such tables must not be confused with representing phenotypes as semantic graphs, because these URIs only have the function of providing semantic links for the E and the Q of an EQ statement from a table to the corresponding ontology classes of ontologies. When an EQ statement is stored as a set of URIs in a table in a relational database, the link between the URI in the E position and its corresponding URI in the Q position is provided implicitly through the position of their cells within the database table, but it is not explicitly stated like it is when representing the EQ statement as a TBox semantic graph. Initially, the EQ format was used for characterizing and classifying different mutant phenotypes of a given model organism by comparing them to their canonical wild-type and then relating them to their underlying genotype [bib_ref] Phenotype ontologies: the bridge between genomics and evolution, Mabee [/bib_ref] [bib_ref] Practical application of ontologies to annotate and analyse large scale raw mouse..., Beck [/bib_ref] [bib_ref] Using ontologies to annotate large-scale mouse phenotype data, Beck [/bib_ref] [bib_ref] Linking human diseases to animal models using ontology-based phenotype annotation, Washington [/bib_ref]. The wild-type functioned as a 'normal' condition and point of reference. Respective phenotype descriptions thus contained comparative phenotype statements [bib_ref] The anatomy of phenotype ontologies: principles, properties and applications, Gkoutos [/bib_ref] that describe instance anatomy in reference to canonical anatomy. However, the EQ approach was soon picked up by evolutionary morphologists, who modified it to describe characters and character states as they are known from phylogenetic character matrices, resulting in direct phenotype descriptions [bib_ref] The anatomy of phenotype ontologies: principles, properties and applications, Gkoutos [/bib_ref] that allow describing phenotypes in the framework of instance anatomy. Typical character and character state descriptions such as "eye color: red" would lend themselves to being translated into EQ statements, with Q representing the character state [bib_ref] Evolutionary characters, phenotypes and ontologies: curating data from the systematic biology literature, Dahdul [/bib_ref] [bib_ref] Phenotype ontologies: the bridge between genomics and evolution, Mabee [/bib_ref] [bib_ref] Connecting evolutionary morphology to genomics using ontologies: a case study from cypriniformes..., Mabee [/bib_ref] [bib_ref] Integrating phenotype ontologies across multiple species, Mungall [/bib_ref]. Many phylogenetic characters, however, cannot be translated into the strict EQ syntax and require modifications [bib_ref] Phenex: ontological annotation of phenotypic diversity, Balhoff [/bib_ref]. Composite characters, for instance, require the reference to more than one entity or quality term, extending the EQ statement to a nested composition, in which the E and/or the Q of the phenotype description are themselves represented as one or several EQ statements. Relational characters require modifying the EQ model to E (QRE) (RE = related entity) and quantitative characters to E (QC) (C = count) (see, e.g., Phenoscape's Guide to Character Annotation). When an EQ statement is stored in a relational database, with the URIs of ontology classes providing only semantic links between the E or the Q of the statement and the definitions of the corresponding ontology classes, additional coding is required to convert the table into an OWL file that documents the EQ statement as a semantic graph. Similarly, EQ statements stored within NeXML files [bib_ref] NeXML: rich, extensible, and verifiable representation of comparative data and metadata, Vos [/bib_ref] , an XML-based phylogenetic data exchange standard inspired by NEXUS (e.g., [bib_ref] Phenex: ontological annotation of phenotypic diversity, Balhoff [/bib_ref] , as used by the Phenoscape project, require separate software for conversion to a semantic model in OWL. Although at first glance the class-based pre-composition and the class-based post-composition approaches appear to be incompatible, it has been demonstrated that they are actually complementary and fully compatible because single term expressions and (composite) EQ statements can be related to each other as being equivalent, providing each pre-composed ontology class with a possible equivalent logical definition in the form of a corresponding EQ statement [bib_ref] Integrating phenotype ontologies across multiple species, Mungall [/bib_ref]. Any given EQ description of a phenotype can thus be translated to the definition of a corresponding ontology class (i.e., Semantic Phenotype expression class) that represents that specific phenotype, and vice versa [bib_ref] Representing Phenotypes in OWL, Mungall [/bib_ref] [bib_ref] Automatically transforming pre-to post-composed phenotypes: EQ-lising HPO and MP, Oellrich [/bib_ref]. As a consequence, EQ statements can be described within ontologies, documented and exported as OWL files, and represented as class-based TBox semantic graphs. Taxonomists became interested in the OWL-based documentation of phenotypes and suggested that the EQ approach could also be used for taxonomically describing phenotypes [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref] [bib_ref] Time to change how we describe biodiversity, Deans [/bib_ref]. Respective EQ statements can be composed as axioms of corresponding ontology classes with the help of ontology editors such asProtégé, using OWL Manchester Syntaxand following general composition schemes [bib_ref] Representing Phenotypes in OWL, Mungall [/bib_ref] [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref] [bib_ref] Folding wings like a cockroach: a review of transverse wing folding ensign..., Mikó [/bib_ref] [bib_ref] Taxonomic Synopsis of the Ponto-Mediterranean Ants of Temnothorax nylanderi Species-Group, Csősz [/bib_ref]. In order to express that a particular specimen bears a specific phenotype, the specimen is represented in OWL as an individual resource with its own URI. This individual is specified to be an instance of the ontology class that defines the phenotype. The resulting direct phenotype descriptions have been called Semantic Phenotypes and each Semantic Phenotype is attached to one (or more) particular specimen [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref] [bib_ref] Time to change how we describe biodiversity, Deans [/bib_ref] [bib_ref] Folding wings like a cockroach: a review of transverse wing folding ensign..., Mikó [/bib_ref] [bib_ref] Taxonomic Synopsis of the Ponto-Mediterranean Ants of Temnothorax nylanderi Species-Group, Csősz [/bib_ref] [bib_ref] Phenotype ontologies: are homology relations central enough? A reply to Deans et..., Franz [/bib_ref]. Semantic Phenotypes, thus, can be completely expressed in OWL and stored in a separate OWL file [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref]. In some sense, Semantic Phenotypes combine the class-based pre-composition approach with the classbased post-composition approach since the description of Semantic Phenotypes first requires the definition of the corresponding phenotype classes (pre-composition), which in turn use ontology classes in their axioms (postcomposition). Direct anatomical phenotype descriptions are essentially 'factual' anatomical descriptions consisting of assertional statements that document empirical observations about particular anatomical entities. A Semantic Phenotype represents the 'factual' anatomical description through a single ABox expression that specifies a phenotype class that is instantiated by the ODU. All the actual descriptive content is implicitly contained in the referenced phenotype class in the form of class axioms and thus TBox expressions. Consequently, the amount of required TBox expressions in the description exceeds the necessary minimum. This represents a conceptual choice that can be traced back to the history of the Semantic Phenotype approach, which applied semantic workflows and tools that originated from mutagenesis research on model organisms (i.e., Homo sapiens and others) and were therefore conceptualized for comparative phenotype descriptions that reference to canonical anatomy. A phylogenetic character statement compared to a phenotype description based on the EQ model. The upper line represents the character statement "eye color: red", following the syntax suggested by Sereno [bib_ref] Logical basis for morphological characters in phylogenetics, Sereno [/bib_ref] , with L 1 indicating the (first) locator and V the variable, which together constitute the character part of a character statement. The character state is represented by the value v 1 , which is one of the possible states defined for the character statement. This phylogenetic character statement can be translated into the EQ statement eye+red, with eye from, e.g., the Mouse Adult Gross Anatomy Ontology (id MA:0000261), representing the locator L 1 part of the character and red from PATO (id PATO:0000322) representing the value v 1 of the character state. Because PATO organizes quality terms in a nested hierarchy of increasingly differentiated attributes, the reference to red implicitly references also color (id PATO:0000020) and thus the variable V part of the character (cf. While this choice has technical implications, as long as phenotypes are only annotated within relational database tables using sets of URIs of ontology classes instead of documenting them as semantic graphs in a knowledge base, the technical implications have no practical consequences. The most important technical consequence of documenting descriptive contents using TBox expressions is the fact that all entities, related entities, and qualities mentioned in the axioms of phenotype ontology classes used in Semantic Phenotypes are anonymous resources and therefore do not possess their own URIs. They cannot be referenced individually, and more complex Semantic Phenotypes cannot be easily partitioned into simpler descriptive fragments. This has far-reaching consequences that also affect reasoning over Semantic Phenotypes, requiring the implementation of additional rules to relate anonymous resources with one another and with the specimen they describe. For instance the phenotype "antenna longer than eye" can be expressed in OWL Manchester syntax as 'has part some (antenna and bearer of some (length and increased in magnitude relative to some (length and inheres in some eye)))' ('and' being used in the sense of intersection of two sets and 'some' in the sense of the existential quantifier 'there exists a' or 'some instance of'), with the consequence that 'some antenna' and 'some eye' are anonymous resources so that "to be an instance of this class, an antenna needs to merely be longer than at least one eye in the world, not necessarily an eye possessed by the same organism" ( [51], p. 643). ## Phenotype knowledge graphs: phenotype descriptions as instance-based abox representations Another approach for using ontologies to standardize direct phenotype descriptions has been suggested that represents particular phenotypes as instance-based ABox semantic graphs, called Phenotype Knowledge Graphs [bib_ref] The linguistic problem of morphology: structure versus homology and the standardization of..., Vogt [/bib_ref] [bib_ref] Learning from Linnaeus: towards developing the foundation for a general structure concept..., Vogt [/bib_ref] [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. Phenotype Knowledge Graphs can be stored in separate OWL files and take the form of 'factual' anatomical descriptions. Contrary to Semantic Phenotypes, Phenotype Knowledge Graphs refer to particulars for the description of a given phenotype and thus minimize the amount of required TBox expressions to the class specifications that each identified part of the ODU instantiates, while describing the particular qualities of the parts and the actual relationships between them as ABox expressions, instead of describing them through class axioms. In other words, each anatomical entity, quality, and property described in a Phenotype Knowledge Graph is represented as a particular that possesses its own URI and that instantiates a corresponding ontology class, which in turn is necessarily specified using TBox expressions. As a consequence, each described part, quality and property can be individually referenced and identified through its own URI. When describing an ODU following the Phenotype Knowledge Graph approach, one first must decompose the ODU into the constituent parts one wants to cover in the description. Each part belongs to a specific kind of anatomical entity and is therefore represented as an instance of the corresponding ontology class. All the described parts are related to one another through parthood relations. The resulting parthood hierarchy provides the organizational backbone for a Phenotype Knowledge Graph and is in that function comparable to the taxonomy (i.e., class-subclass hierarchy) of classes of an ontology [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. Next, one can describe each constituent part in more detail, specifying its various properties and qualities, including the specification of relations between parts . By referencing the ontology classes that are instantiated by described parts, Phenotype Knowledge Graphs link to one or more ontologies, which allows applications that process and analyze Phenotype Knowledge Graphs to utilize not only the information contained in the descriptions themselves (instance anatomy data) but also the information contained in all referenced ontologies (canonical anatomy and thus invariant knowledge). The combined information can be used for inferencing and quantitatively comparing different Phenotype Knowledge Graphs [bib_ref] Assessing similarity: on homology, characters and the need for a semantic approach..., Vogt [/bib_ref] [bib_ref] The logical basis for coding ontologically dependent characters, Vogt [/bib_ref] [bib_ref] Towards a semantic approach to numerical tree inference in phylogenetics, Vogt [/bib_ref]. Phenotype Knowledge Graphs can be meaningfully fragmented into several flexibly manageable subgraphs, with each subgraph corresponding to a specific type of descriptive statement [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. For example, the parthood relation between two anatomical entities, the shape specification of a particular anatomical structure, or the specification of its weight measured in milligrams can be associated with its own named graph resource . A named graph resource is a URI that identifies a set of triple statements by adding this URI to each triple belonging to the named graph, thus turning the triples into quads. Phenotype Knowledge Graphs can be organized into named graphs, stored in a tuple store, and be made accessible through a SPARQL 12 endpoint. Phenotype Knowledge Graphs express phenotype descriptions wherever possible as assertional statements in the form of instance-based ABox semantic graphs, requiring only a minimum amount of TBox expressions. Consequently, the Phenotype Knowledge Graph approach does not suffer from the technical implications resulting from expressing phenotype descriptions as universal statements using the EQ format. In the following, we discuss these technical implications and evaluate their practical consequences based on the technical requirements for empirical data mentioned above and their relation to eScience-compliant data and metadata standards and the FAIR Guiding Principles. # Discussion Before we discuss the technical advantage of the instance-based ABox approach and its practical implications, we want to emphasize once more that the here discussed limitations of the TBox approach apply in the context of documenting empirical data and metadata in a knowledge graph. There are many other contexts, in which TBoxes can be superior to ABoxes. For instance, when documenting or using invariant knowledge (see 104) and thus universal statements instead of assertional statements, where ABoxes cannot be used. In anatomy, this would relate to the context of canonical anatomy. When reasoning over your data is important, TBoxes may in some cases also be superior to ABoxes. However, whereas, reasoning has primarily been applied for validating the consistency of class hierarchies and for inferring additional subsumption relationships [bib_ref] Use of OWL within the gene ontology, Mungall [/bib_ref] , the need for reasoning over ABoxes has been identified by now and corresponding reasoners such as Arachne [bib_ref] Arachne: an OWL RL reasoner applied to gene ontology causal activity models..., Balhoff [/bib_ref] are being developed that support reasoning on, e.g., property relationships. Reasoners such as ELK, that are commonly used with TBoxes, use the OWL EL profile, which does not support ABox reasoning very well. Arachne uses the OWL RL profile, which is better suited for instance data. Arachne can, e.g., be used when adding an ABox to a knowledge graph for suggesting additional inferred statements and for checking for consistency in real time-TBox reasoners such as ELK are well suited for tasks like ontology classification and consistency checking of ontology classes, but do not perform well for real-time multi-user online systems focused on ABox graphs, because they do not support axioms like inverse properties, property ranges, and materialization of object property assertions [bib_ref] Arachne: an OWL RL reasoner applied to gene ontology causal activity models..., Balhoff [/bib_ref]. When having to compare an actual state of a system, as it can be recorded, e.g., via sensors and documented as an ABox, against a target state, which could be an established standard documented as a Phenotype Knowledge Graph. The instance-based ABox semantic graph shows the description of a multicellular organism. It consists of instances (purple-bordered boxes), each of which instantiates a specific ontology class (yellow-bordered boxes with rounded corners) through the type property. All instances referring to anatomical entities possess a human-readable label (grey-bordered box connected through the label property) and are connected via parthood relations, forming a partonomy. The partonomy indicates that this instance of multicellular organism possesses an instance of insect head that, in turn, possesses an instance of antenna. The multicellular organism instance is further described to have a flattened shape and a measured live weight of 84.3 milligrams. The semantic graph is organized and fragmented into different subgraphs, each of which is contained in its own named graph (dashed-bordered colored boxes). Each subgraph contains information relating to a specific perceptual question that can only be answered empirically. In other words, each named graph contains a separate empirical observation. And each named graph also possesses its own URI and instantiates a named graph ontology class. The Phenotype Knowledge Graph is the union of all the named graphs TBox, you can check the ABox for consistency against the TBox using Arachne. [bib_ref] Strengths and limitations of formal ontologies in the biomedical domain, Schulz [/bib_ref] Due to the tabular architecture of relational databases, TBoxes have an advantage over ABoxes when storing data in a relational database, because assertional statements can be documented as instances of ontology classes that, in turn, follow the EQ or EAV model and provide the description of the actual content in their class axioms. Therefore, one only has to store the URI of the ontology class as a value in a respective table to document the content specified through that class's axioms. The choice of whether to use a relational database or a knowledge graph for storing, documenting, and managing research data should be driven by the requirements of your study or project and the competency questions that you derive from your respective user stories. Relational databases are well suited for closed world systems, for which you can specify the data schema before populating your database with data, whereas knowledge graphs are well suited for open world systems and thus systems that assume incomplete knowledge by default, where you can easily extend the data schema on-the-fly. Also, (i) if the query structure is well known and expected to be stable-you know, which questions the dataset has to answer and these questions will not likely change in the future, (ii) if you know that the dataset may grow, but only the same type of data will be added, or (iii) if your dataset is not complex and its data points are not heavily interconnected so that it can be easily represented in the tabular structure of a relational database, relational databases may be superior to knowledge graphs as a technical solution for your data management. In the following, we discuss the technical difference between semantic phenotypes and phenotype knowledge graphs as examples for the class-based TBox and the instance-based ABox approach and the practical implications of this difference in the context of documenting empirical data and metadata in a knowledge graph. ## Decomposing phenotype descriptions into separate observation-based statements Unlike Semantic Phenotypes, Phenotype Knowledge Graphs can be fragmented in various ways into meaningful subgraphs. As a consequence, they provide significantly more flexibility in what can be done with them. Each subgraph can be organized in its own particular named graph that possesses its own URI (see . Each named graph resource can be associated with a corresponding ontology class that it instantiates. These classes can be defined in a domain reference ontology for anatomy that specifies a semantic data model for anatomy [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. In this way, one could define an ontology class for each type of descriptive statement relevant for phenotype descriptions. Each class defined this way can be understood to correlate with a specific perceptual question that can only be answered by studying the relevant parts of the given ODU. The respective question thereby functions like a perceptual category that is part of a general phenotype structure concept [bib_ref] The linguistic problem of morphology: structure versus homology and the standardization of..., Vogt [/bib_ref] [bib_ref] Learning from Linnaeus: towards developing the foundation for a general structure concept..., Vogt [/bib_ref]. Examples for such questions would be: What is the weight of this anatomical structure? What is the length of this anatomical line? What is the volume of this anatomical space? What is the position of this anatomical point? What is the color of this anatomical surface? What is the general shape of this anatomical structure? What is the biological function of this anatomical structure? From which structure did this anatomical structure develop? Each named graph belonging to a phenotype description refers to the combination of (i) a particular part of the ODU and (ii) a specific perceptual category. Fragmenting a given phenotype description into several such named graphs can be understood as the decomposition of the description into its smallest units of empirical information and thus into a set of particular descriptive statements. As a consequence, any given Phenotype Knowledge Graph can be fragmented into its descriptive statements in the form of subgraphs and these subgraphs can be united again to return the Phenotype Knowledge Graph. This general approach is not restricted to anatomy and can be applied to any empirical data. The decomposability of Phenotype Knowledge Graphs in particular and of instance-based ABox semantic graphs in general is the most important technical difference compared to Semantic Phenotypes and class-based TBox semantic graphs and has significant consequences that substantially affect various practical aspects. ## The explorability of phenotype descriptions Based on the ontology classes of descriptive named graphs discussed above, one can flexibly define various data views for exploring Phenotype Knowledge Graphs [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. Each data view is defined in reference to one or more such classes. One data view could, for instance, be defined in reference to the class of weight measurements, whereas another one could comprise all classes that contain measurements in general. Applying the former data view on a given description would result in the union of all subgraphs of the description that contain weight measurement data, whereas the application of the latter would result in the union of all subgraphs containing measurement data in general. The definition of various such data views would significantly improve the possibility to meaningfully navigate semantic graphs of phenotype descriptions without users of respective applications having to write deeply nested SPARQL queries, because only the corresponding named graphs must be identified. This, again, applies in general to all kinds of empirical data that are represented as instance-based ABox semantic graphs. Unfortunately, Semantic Phenotypes and any other class-based TBox semantic graph cannot be fragmented this way, because the entities, related entities, and qualities that class axioms refer to are anonymous resources and thus cannot be individually referenced and identified (see above). Therefore, Semantic Phenotypes cannot be explored to the same degree as Phenotype Knowledge Graphs. ## Linking relevant metadata and supplementary contents to phenotype descriptions Metadata are statements about statements. In the case of phenotype descriptions, metadata refer to who contributed which parts of the description, based on which evidence, and using which instruments, where and when (see [fig_ref] Figure 4: Metadata about a Phenotype Knowledge Graph [/fig_ref]. Modeling statements about statements within OWL/RDF is not trivial and various approaches have been suggested [bib_ref] Evaluation of metadata representations in RDF stores. Ngonga Ngomo A-C, Fundulaki I,..., Frey [/bib_ref]. OWL itself provides the possibility to make statements about statements using standard reification, by specifying the statement about which one wants to make statements through three additional triple statements (i.e., statement_URI subject subject_ URI; statement_URI predicate predicate_URI; state-ment_URI object object_URI). While this may be a practical solution for making statements about a single triple statement, it becomes very impractical if one has to make statements about a subgraph that consists of several triple statements (see example [fig_ref] Figure 4: Metadata about a Phenotype Knowledge Graph [/fig_ref]. For such cases, the use of named graphs is a good choice. Moreover, named graphs also outperform other metadata representation models when conducting more complex queries [bib_ref] Evaluation of metadata representations in RDF stores. Ngonga Ngomo A-C, Fundulaki I,..., Frey [/bib_ref]. Because each descriptive statement belonging to a Phenotype Knowledge Graph is organized in its own particular named graph and this named graph has its own URI, it can be individually referenced for associating relevant metadata information to it, such as on which specimen the observation is based, which microscope has been used or the literature source from which the information in that subgraph of the description has been taken and how reliable that source is [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. Each such metadata, in its turn, can be documented in its own named graph and thus be clearly separated from the actual description. The combination of a particular descriptive named graph and its associated metadata named graph can be published separately from the whole description as a nano-publication [bib_ref] Nano-Publication in the e-science era Nano-Publication in the e-science era, Mons [/bib_ref] [bib_ref] The anatomy of a nano-publication, Groth [/bib_ref] [bib_ref] Decentralized provenance-aware publishing with nanopublications, Kuhn [/bib_ref]. with its associated metadata. The Phenotype Knowledge Graph is organized into three subgraphs (I, II, and III), each of which has its own set of metadata statements associated. When compared to , one can identify the three subgraphs in reference to named graphs: subgraph I) refers to the 'shape named graph', subgraph II) to the 'weight measurement named graph', and subgraph III) to the union of the two 'parthood named graphs' Moreover, by referring to the URI of the particular named graph, one can also link natural language descriptions and semantically annotated media contents to each descriptive statement, as well as comments and other annotations. And because each described part, quality, and property possesses its own URI in a Phenotype Knowledge Graph, images can be annotated with regions of interest using these URIs to indicate that they depict a particular part, quality or property, which is not possible with Semantic Phenotypes. As a consequence, the use of the description named graphs allows for differentially assigning metadata, unstructured natural language texts, and media contents at the level of smallest units of semantically meaningful empirical information contained in a Phenotype Knowledge Graph instead of having to assign them to the description as a whole, and this information can be published as a micro-publication [bib_ref] Micropublications: a semantic model for claims, evidence, arguments and annotations in biomedical..., Clark [/bib_ref]. And again, this is not restricted to the domain of anatomy, but can be applied to all kinds of empirical data that are represented as instance-based ABox semantic graphs. Unfortunately, Semantic Phenotypes and any other class-based TBox semantic graph cannot be fragmented this way and thus assigning metadata, natural language texts, and media contents at the level of smallest units of empirical information is not that straight forward. ## Expandability of phenotype descriptions It is impossible to describe a given specimen covering all aspects that could be relevant. Like any other description of a particular material entity or process, each phenotype description represents a decomposition that is based on a virtual partition of the ODU into the parts that are relevant for the specific frame of reference applied by the person making the description [bib_ref] Fiat or Bona fide boundary -a matter of granular perspective, Vogt [/bib_ref] [bib_ref] Levels and building blocks-toward a domain granularity framework for the life sciences, Vogt [/bib_ref]. Due to the phenotypic complexity of anatomical entities, which often covers several levels of granularity, ranging from the molecular level to the cellular level and the level of gross anatomy, descriptions of specimens are never complete, irrespective of the applied frame of reference. This applies to Semantic Phenotypes in the same way as to Phenotype Knowledge Graphs. The problem of the incompleteness of phenotype descriptions, however, confronts the Semantic Phenotype approach and the class-based TBox semantic graphs in general with a conceptual dilemma. If a given Semantic Phenotype must be complemented with additional information, resulting in a more detailed representation of the described phenotype, one can choose between: (1) Defining a new phenotype class that incorporates all information of the original phenotype class and, additionally, also covers the new information. The new phenotype class then replaces the original class and the new Semantic Phenotype the original Semantic Phenotype. This, however, would not only result in increasingly complex axiom expressions, which become increasingly incomprehensible, but tracking provenance and all relevant metadata across the different versions will be problematic as well, especially since Semantic Phenotypes cannot be easily fragmented. (2) Defining a new phenotype class that only covers the additional information. The corresponding Semantic Phenotype would complement the original Semantic Phenotype. This is also problematic since the parts and properties mentioned in the class axiom of the original phenotype class cannot be referenced in the complementing phenotype class, because they are anonymous resources. As a consequence, the complementing Semantic Phenotype will, for instance, describe in more detail one of the parts mentioned in the class axiom of the original phenotype class, but the original and the complementing Semantic Phenotype graphs will not connect due to the anonymity of the described parts. Phenotype Knowledge Graphs and instance-based ABox semantic graphs in general, on the other hand, can easily be expanded with additional information. Because each described part, property, and quality possesses its own URI, existing descriptions can be easily expanded through nano-publications and their corresponding metadata be tracked independently of the metadata of the original description. Integrating different frames of reference in a phenotype description As mentioned above, any given phenotype can be described from different frames of reference, e.g., from a purely spatio-structural, a functional, or a developmental perspective. Each frame of reference will likely virtually partition the underlying ODU in its own particular way. Descriptions of the same phenotype that are based on different frames of reference thus often result in incongruent partitions [bib_ref] Levels and building blocks-toward a domain granularity framework for the life sciences, Vogt [/bib_ref]. As a consequence, the representation of a phenotype through a single phenotype ontology class will make it very difficult to cover all information relevant to the various frames of reference relevant in the life sciences because the corresponding class axiom can only model one of the many possible virtual partitions. In other words, a purely spatio-structural description of a given phenotype must be represented with a different phenotype class then a functional, a developmental, or an evolutionary description of that same phenotype. This would result in a spatio-structural Semantic Phenotype, a functional Semantic Phenotype, a developmental Semantic Phenotype, and an evolutionary Semantic Phenotype, each of which would refer to the same given ODU. Due to the problem of anonymous resources, even if each of these descriptions would refer to the same part in the ODU, the resulting graphs would not connect because this part would be represented as anonymous resources. With the Phenotype Knowledge Graph approach, on the other hand, any given phenotype can be described in reference to a specific frame of reference and the resulting graph will connect spatio-structural descriptions of a given described part with its functional, developmental, and evolutionary descriptions, because this part possesses its own URI and thus can be referenced in any possible virtual partition of a given ODU. Contrary to the Phenotype Knowledge Graph approach, the Semantic Phenotype approach with its class axioms seems to be not well suited for integrating different frames of reference in a given phenotype description. ## The open world assumption and the need for negations and specifications of quantities of parts No ODU can be comprehensively described across all possible frames of reference, scales, and granularity levels. No semantic representation of an ODU can be exhaustive in that respect. Any ODU possesses a virtually infinite number of possible partitions so that no phenotype description can be considered to cover all of them. This situation is dealt with by the so-called Open World Assumption (OWA). OWA assumes incomplete information by default. A direct consequence of OWA is that the lack of knowledge about a fact does not immediately imply knowledge of the negation of that fact. This means, for instance, that when a description does not state that a particular insect head has cells as its parts, we cannot conclude that the head is not composed of cells. OWL and description logics-based ontologies adhere to OWA by default, and so do both the Phenotype Knowledge Graph and the Semantic Phenotype approach. In both approaches, when starting to describe an ODU, everything is considered to be possible. This space of possibilities becomes more and more constrained and restricted with the addition of information. Following this notion, phenotype descriptions restrict what is possible [bib_ref] The logical basis for coding ontologically dependent characters, Vogt [/bib_ref]. OWA is not problematic for phenotype descriptions per se. It for instance allows reusing and extending phenotype descriptions, adding more information to already existing descriptions whenever necessary. But in some cases, we want to make clear that a given ODU possesses, e.g., only two antennae and lacks an ovipositor-information that cannot be provided by describing only two antennae and not describing any ovipositor. While one could introduce specific properties to model such information as instance-expressions (see , top, and , any such model will not be compliant with description logics and could therefore not be reasoned on. Making these expressions machine-actionable would thus require additional efforts. Alternatively, one can describe this type of information with the help of class-expressions and thus TBox expressions, using OWL Manchester Syntax. The observation "insect abdomen lacks an ovipositor" translates to the Manchester expression 'not ( has part some ovipositor )' and the observation "insect head has part exactly 3 ocelli" to 'has component exactly 3 ocellus'. 14 Both Manchester expressions can be represented as class-based semantic graphs and be used within the Semantic Phenotype approach as well as the Phenotype Knowledge Graph approach (see , bottom, and . ## Demarcating units of description Another problem with Semantic Phenotypes is whether a given specimen should be described using a single complex Semantic Phenotype or a set of multiple Semantic Phenotypes. Should a phenotype be defined in a single phenotype ontology class or in several such classes? Should the unit of description equal the smallest unit of semantically meaningful empirical information? In the end, it is the question of what is the criterion for demarcating units of description [bib_ref] The linguistic problem of morphology: structure versus homology and the standardization of..., Vogt [/bib_ref] ? And again, part of the problem with Semantic Phenotypes and class-based TBox semantic graphs is the anonymity of the resources referenced in their class axioms. If you want to describe a given ODU using several Semantic Phenotypes, the entities, related entities, and qualities mentioned in the axioms of phenotype classes of different Semantic Phenotypes do not relate to each other, although they may actually refer to the same real entities, because they cannot be individually referenced and identified through the information provided by the graph. This is not the case with Phenotype Knowledge Graphs and instancebased ABox semantic graphs in general because each described part and property possesses its own URI and thus can be referred to in several different graphs. ## Correcting mistakes in phenotype descriptions Researchers are human beings, and human beings make mistakes. Therefore, phenotype descriptions should allow for effective ways to correct for mistakes and thereby unambiguously track what information has been changed and ideally document that change in RDF as well. And again, because Semantic Phenotypes cannot be [bib_ref] Formal ontologies in biomedical knowledge representation, Schulz [/bib_ref] has component is used instead of has part because the latter is a transitive object property and OWL does not permit cardinality constraints to be used in combination with transitive object properties. easily fragmented and the particular parts, properties, and qualities referenced in class axioms do not possess their own URIs, explicitly tracking what information has been changed between the original Semantic Phenotype and the corrected version of that Semantic Phenotype, and documenting in RDF all the changes that have been made, is rather difficult to accomplish. Phenotype Knowledge Graphs, in contrast, can be easily corrected for mistakes. Because the descriptive statements of Phenotype Knowledge Graphs are organized into different named graphs, one can easily correct information in one of them and track provenance and relevant metadata for it, as well as document in the metadata all changes that have been made. ## Universal usability and reusability of phenotype descriptions Being able to fragment a Phenotype Knowledge Graph into smaller subgraphs allows using only those parts of the data that are relevant for a given research question, while ignoring all parts that are irrelevant. The differentiation of types of observation and the modelling of respective data into corresponding named graphs allows meaningful fragmentation of data and reuse in various frameworks. While this is in principle also possible with Semantic Phenotypes, the extraction of only the relevant data is not as straightforward. ## Generation of phenotype descriptions As already mentioned above, in order to generate a Semantic Phenotype, the corresponding phenotype must be first defined as an ontology class before the description itself can be generated, which in turn only specifies that a given ODU instantiates that specific class. Technically, the actual phenotype description is contained in the definition of the ontology class. Defining such phenotype ontology classes is usually conducted using OWL Manchester Syntax, which can become very complex, especially if the underlying phenotype is complex and the description fine-grained. For instance the EQ statement "head color: reddish brown, except for dark brown to black postgena, occiput, vertex; mandibles, maxillary and labial palps yellowish; scape, pedicel, F1 and F2 yellow, subsequent flagellomeres progressively darker" translates to the OWL Manchester Syntax expression (example taken from suppl. Material 2 of [bib_ref] New Dryocosmus Giraud species associated with Cyclobalanopsis and non-Quercus host plants from..., Tang [/bib_ref] : has part some ( head and ((not ( clypeus )) and (not ( mandible and ((((not ( antenna )) and ( bearer of some red )) and (not ( labial palp ))) and (not ( maxillary palp )))))) and ( has part some ( labial palp and ( bearer of some yellow ))) and ( has part some ( mandible and ( bearer of some yellow ))) and ( has part some ( maxillary palp and ( bearer of some yellow ))) and ( has part some ( occiput and ( bearer of Two alternative models for documenting absences using the instance-based ABox semantic graph approach. Within the Phenotype Knowledge Graph approach, the observation "insect abdomen lacks an ovipositor" can be modeled in two alternative ways. Top: Shows a representation of the observation using only ABox expressions. This requires the introduction of the object property has not part any that has an instance as a domain restriction and a class as range restriction. This is not part of the OWL syntax and would require the introduction of additional tools for making it machine-actionable. Bottom: Shows a representation of the observation using a combination of ABox and TBox expressions. The instance of insect abdomen instantiates not only the class insect abdomen but also the class absent ovipositor phenotype, which is characterized as the complement class of the class of entities that have some ovipositor as their part. This description is compliant with description logics and is directly machine-actionable. Purple-bordered box = instance resource; yellow-bordered box with rounded corners = ontology class resource; grey-bordered box with rounded corners = anonymous class; blue-bordered octagon = object property class; labeled arrow = property resource some dark brown ))) and ( has part some ( pedicel and ( bearer of some yellow ))) and ( has part some ( postgena and ( bearer of some dark brown ))) and ( has part some ( scape and ( bearer of some yellow ))) and ( has part some ( vertex and ( bearer of some dark brown ))) and ( has part some ( first flagellomere and ( bearer of some yellow ))) and ( has part some ( second flagellomere and ( bearer of some yellow ))) and ( has part some ( fifth flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some sixth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( third flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some fourth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( fourth flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some fifth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( sixth flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some seventh flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( seventh flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some eighth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( eighth flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some ninth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( ninth flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some Two alternative models for documenting exact counts of parts using the instance-based ABox semantic graph approach. Within the Phenotype Knowledge Graph approach, the observation "insect head has part exactly 3 ocelli" can be modeled in two alternative ways. Top: Shows a representation of the observation using only ABox expressions. This requires modeling the parthood relation as a directed relational quality and as a consequence of that the introduction of an object property towards class that has an instance as a domain restriction and a class as range restriction. Unfortunately, modeling the observation this way is not compliant with description logics and would require the introduction of additional tools for making it machine-actionable. Bottom: Shows a representation of the observation using a combination of ABox and TBox expressions. The instance of insect head instantiates not only the class insect head but also the class exact ocellus count phenotype, which is characterized as a cardinality restriction on a combination of a property and a class. This description is compliant with description logics and is directly machine-actionable. Purple-bordered box = instance resource; yellow-bordered box with rounded corners = ontology class resource; grey-bordered box with rounded corners = anonymous class; grey-bordered box with sharp corners = value; blue-bordered octagon = object property class; labeled arrow = property resource color brightness and ( inheres in some tenth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( eleventh flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some twelfth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( twelfth flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some thirteenth flagellomere )))) and ( bearer of some light brown ))) and ( has part some ( thirteenth flagellomere and (( bearer of some color brightness ) and ( increased in magnitude relative to some ( color brightness and ( inheres in some flagellomere 14 )))) and ( bearer of some light brown )))). [bib_ref] On the importance of standardisation in life sciences, Brazma [/bib_ref] Obviously, respective class axioms can consist of many levels of nested expressions organized in parentheses, which many researchers have a hard time to read and comprehend. Also, this method of description is very error-prone due to this nested syntax. Alternatively, such OWL Manchester Syntax based expressions can be restricted to a certain threshold of slots. In Phenoscape, for example, templates are used with three slots. Restricting the descriptions to three slots keeps Semantic Phenotypes from getting too complicated, but also prevents them from being as precise and detailed as possible. Another problem with the Semantic Phenotype approach becomes apparent when considering morphometric data. [bib_ref] Minimum information about a microarray experiment (MIAME)-toward standards for microarray data, Brazma [/bib_ref] When describing phenotypes based on a set of multiple measurements, the Semantic Phenotype approach would require for every possible combination of measurements the definition of a corresponding phenotype class. With the addition of more quantitative properties, this would result in exponentially increasing numbers of possible phenotype classes. Documenting every type of measurement as a single Semantic Phenotype somewhat mitigates the problem but results in the above-mentioned problem of disconnected information due to anonymous resources. Whereas the generation of Phenotype Knowledge Graphs does not face these problems, it requires the development of an adequate application that allows researchers describing phenotypes respectively. This application could utilize the hierarchical structure of parthood relations between described parts of a given description to organize its interface. For each description, the partonomy could be visualized as a tree-like structure of described parts. This partonomy could also function as a navigator for selecting a particular described part. Each part, in turn, has its own input form associated with it that allows a detailed description of that part and can be accessed by selecting the part within the partonomy. We are currently developing such an application for the online anatomical data repository Morph D Baseand a functional prototype is available. The interface has been developed in close cooperation with several anatomyexperts from different backgrounds, who served as use-cases during its development. They considerably contributed to it, allowing an intuitive generation of Phenotype Knowledge Graphs. All data is stored in a Jena tuple store and descriptions are organized into several description named graphs as described above. The interface provides a human-readable HTML-version of the description while retaining a machineactionable and reasoning-capable version that can be accessed through a SPARQL endpoint, thus allowing exploiting semantic technology to its full potential and offering Phenotype Knowledge Graphs as Linked Open Data. ## Potential suitability of abox and tbox semantic graphs for data and metadata standards In times of eScience, a standard for data and metadata must cover machine-actionability regarding terminological aspects relating to concepts (meaning) and nomenclature (reference) and assertional aspects relating to formats (syntax and file format) and contents (data model) [bib_ref] eScience and the need for data standards in the life sciences: in..., Vogt [/bib_ref] [bib_ref] The need for data standards in zoomorphology, Vogt [/bib_ref] [bib_ref] Signs and terminology: science caught between language and perception, Vogt [/bib_ref] (see [fig_ref] Table 1: Potential suitability of TBox and ABox semantic graphs for meeting eScience-compliant data... [/fig_ref]. Moreover, it must also comply with the FAIR Guiding Principles [bib_ref] The FAIR guiding principles for scientific data management and stewardship, Wilkinson [/bib_ref] [bib_ref] Interoperability and FAIRness through a novel combination of Web technologies, Wilkinson [/bib_ref] [bib_ref] Evaluating FAIR Maturity Through a Scalable, Automated, Community-Governed Framework, Wilkinson [/bib_ref] (see [fig_ref] Table 2: Potential suitability of TBox and ABox semantic graphs for meeting the FAIR... [/fig_ref]. An eScience-compliant concept standard requires a machine-and human-readable specification of the meaning of all concepts used in data and metadata statements. The specification provides information about what we know of the corresponding real universal, i.e., the kind. Semantic Phenotypes and Phenotype Knowledge Graphs both comply with this by referencing ontology terms that, in turn, provide unambiguous definitions of meanings for concepts both in human-and machine-readable ways. The nomenclatural standard requires unambiguous specification of the reference of the words, symbols, and IDs used in data and metadata statements. It provides an unambiguous link between term and concept. Again, Semantic Phenotypes and Phenotype Knowledge Graphs both comply with this standard by using machinereadable persistent URIs in addition to human-readable labels for referring to ontology classes. The link between word, symbol, or ID and its corresponding concept, which in turn provides the meaning, is thus clear and unambiguous. This allows the reuse of ontology terms in any semantic graph without the necessity to include the entire ontology specification. However, only Phenotype Knowledge Graphs provide this standard also for all parts and properties mentioned in the description, which Semantic Phenotypes only reference anonymously. The combination of concept and nomenclatural standard covers the terminology-related aspects of an eScience-compliant standard and ensures that phenotype descriptions are semantically transparent, allowing even non-experts to understand and interpret them correctly. In addition to these terminology-related aspects, eScience-compliant data and metadata standards must also cover assertions-related aspects, which is covered by a combination of a format and a content standard that ensures that phenotype descriptions are comparable, reusable, computer-parsable, and communicable through the Web. The format standard requires a machine-readable specification of the syntax and file format to be used when documenting, storing, communicating, and processing data and metadata statements on the Web. Semantic Phenotypes and Phenotype Knowledge Graphs provide this through the possibility to store the respective semantic graphs in OWL files, which can be serialized to RDF. As a consequence, Semantic Phenotypes and Phenotype Knowledge Graphs both provide a basic level of findability, accessibility, and explorability because they can take the form of semantic graphs and any semantic graph can be searched using SPARQL. The query pattern of a SPARQL query is itself represented as a semantic graph that may contain variables and wildcards. The main mechanism of a SPARQL query is matching the query pattern with the semantic graph to be queried. A repository for Semantic Phenotypes or Phenotype Knowledge Graphs stored in a tuple store would allow searching for descriptions of heads of a specific taxonomic group that possess a specific type of antenna and that have a weight larger than 10 mg and retrieve a list of corresponding phenotype descriptions. Regarding querying semantic graphs, however, it is important to note that querying TBox expressions is more difficult than querying ABox expressions. In case the graph contains class definitions in the form of axioms expressed in OWL, the basic graph-pattern-matching of SPARQL must be defined using entailment regimes. Querying under entailment regimes is more complex and computationally difficult under full expressivity of OWL [bib_ref] Optimizing SPARQL query answering over OWL ontologies, Kollia [/bib_ref] [bib_ref] Ontological query answering over semantic data, Stamou [/bib_ref]. As a consequence, querying Phenotype Knowledge Graphs is more straight forward and computationally less difficult than querying Semantic Phenotypes. In ABox semantic graphs, we can associate a specific content standard for each descriptive named graph class. The content standard specifies the general structure of how to express the corresponding type of empirical information in terms of RDF triples by defining a (meta) data include qualified references to other (meta)data corresponding semantic graph pattern [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref] , for example using an RDF graph schema language such as SHACL or ShEx. The same can be applied using TBox semantic graphs for standardizing the definitions of ontology classes. When applied consistently throughout a data repository that stores and manages phenotype descriptions, the set of templates would specify a semantic model for phenotype data and metadata [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. Such data and metadata models would not only complement the format standard by further specifying the syntax of all types of descriptive and metadata statements relevant for phenotype descriptions but also specify the content standard aspect of eScience-compliant standards [bib_ref] eScience and the need for data standards in the life sciences: in..., Vogt [/bib_ref] [bib_ref] The need for data standards in zoomorphology, Vogt [/bib_ref] [bib_ref] Signs and terminology: science caught between language and perception, Vogt [/bib_ref]. The content standard requires the specification of which information is relevant for a specific type of data or metadata statement and provides a basic categorization and classification of possible contents belonging to a given domain and the corresponding schemata for modeling and documenting them. The graph pattern associated with each descriptive named graph class specifies which information must be provided for the given type of data statement. The same can be done with metadata statements, which should be associated with their own particular named graphs too. This significantly increases the comparability of Semantic Phenotypes and Phenotype Knowledge Graphs [bib_ref] Organizing phenotypic data-a semantic data model for anatomy, Vogt [/bib_ref]. Whereas the specification of templates for class axioms guarantee a certain level of comparability between different Semantic Phenotypes [bib_ref] A semantic model for species description applied to the ensign wasps (Hymenoptera:..., Balhoff [/bib_ref] , these templates are very general and not customized to basic perceptual categories such as the semantic graph templates associated with Phenotype Knowledge Graphs. Therefore, Semantic Phenotypes are not to the same degree comparable with each other as Phenotype Knowledge Graphs. Regarding the FAIR Guiding Principles, Phenotype Knowledge Graphs are slightly superior to Semantic Phenotypes with respect to the findability, accessibility, and interoperability criteria mentioned by Wilkinson et al. [bib_ref] The FAIR guiding principles for scientific data management and stewardship, Wilkinson [/bib_ref] (see [fig_ref] Table 2: Potential suitability of TBox and ABox semantic graphs for meeting the FAIR... [/fig_ref]. With respect to the criterion of reusability, the way phenotype data are represented is rather irrelevant, and it is more a question of implementation within an application and the quality of the metadata provided by the creators of a given phenotype description. Anyhow, what both representations of phenotypes lack is good human-readability of their data and associated metadata. This is a general problem with semantic graphs: whereas their machine-actionability can be excellent, their human-readability is usually poorhumans neither want to read RDF/OWL files nor triple statements or complex graphs. Moreover, since machines have problems with fuzzy and context-dependent information-something typically found in natural language texts-semantic graphs tend to be more complex and explicit than human readers need, adding information that human readers distract from the information they are interested in. Ideally, applications storing data in the form of semantic graphs feature tools that translate semantic graphs into human-readable statements that can be presented, for instance on an HTML page. ## Machine-actionability of phenotype descriptions As already discussed above, both Semantic Phenotypes and Phenotype Knowledge Graphs are machineactionable. However, because each particular descriptive statement and described part, property, quality, and relation in a Phenotype Knowledge Graph possesses its own URI and reasoning over instance-based ABox semantic graphs is computationally less difficult than reasoning over class-based TBox semantic graphs, the machineactionability of Phenotype Knowledge Graphs allows for broader practical applicability. Algorithms can use the information contained in a given set of Phenotype Knowledge Graphs together with the information contained in all ontologies they reference. By traversing the parthood hierarchy of a Phenotype Knowledge Graph and the class-subclass hierarchy of referenced ontologies, algorithms could match and map nodes between different Phenotype Knowledge Graphs and align them, in order to identify units of comparison between them and measure the overall degree of similarity between them [bib_ref] Assessing similarity: on homology, characters and the need for a semantic approach..., Vogt [/bib_ref]. Results of respective comparisons could themselves be documented as for instance separate consensus Phenotype Knowledge Graphs [bib_ref] Assessing similarity: on homology, characters and the need for a semantic approach..., Vogt [/bib_ref] that supplement the originally compared Phenotype Knowledge Graphs. The ability to measure the degree of similarity between a particular Phenotype Knowledge Graph and all Phenotype Knowledge Graphs stored in a phenotype repository would also greatly facilitate searching across phenotype descriptions, resulting in a search # Conclusion Class-based and instance-based semantic representations of phenotypes are both overall FAIRer than phenotype descriptions in the form of unstructured natural language texts, especially regarding their machineactionability. By linking URIs to corresponding ontology class definitions, they both provide unambiguous links to the meaning of the terms used in the descriptions and therewith provide the much-needed semantic transparency. This allows researchers to understand the descriptions, independent of their backgrounds within the life sciences and their expertise with the particular anatomy of the respective taxon. Moreover, when stored in adequate repositories, phenotype descriptions in the form of Semantic Phenotypes and Phenotype Knowledge Graphs become findable and accessible. Due to their use of URIs, searching a repository for specific phenotype data becomes possible. Searching for specific phenotype data in published literature, in contrast, is not only tedious and exhausting but significantly less efficient and often also hampered by pay-walls. The incomprehensibility of phenotype descriptions for non-experts and their limited findability and accessibility has been one of the most detrimental problems of anatomy/morphology as a discipline in academia. If colleagues from other disciplines have problems finding your data and when they find them, they have problems understanding them, they will likely think twice to collaborate with you and are therefore less interested in your research. Both the Semantic Phenotype approach and the Phenotype Knowledge Graph approach have the potential to change this. Moreover, both approaches enable the application of machine-reasoning, which can be utilized for various analytical purposes, for inferencing, and for checking the consistency of the data [bib_ref] Arachne: an OWL RL reasoner applied to gene ontology causal activity models..., Balhoff [/bib_ref] [bib_ref] Toward synthesizing our knowledge of morphology: using ontologies and machine reasoning to..., Dececchi [/bib_ref] [bib_ref] Using AberOWL for fast and scalable reasoning over BioPortal ontologies, Slater [/bib_ref] [bib_ref] Reasoning over anatomical homology in the Phenoscape KB, Mabee [/bib_ref]. However, with respect to the FAIR Guiding Principles and their suitability for meeting eScience-compliant standards, the Phenotype Knowledge Graph approach is superior to Semantic Phenotypes, because querying its graphs is computationally less difficult and integrating metadata straight forward. Apart from that, looking at the various practical implications of the technical differences between the two general approaches, the instance-based ABox semantic graphs approach seems to be in general superior to the class-based TBox semantic graphs approach in the context of documenting and managing empirical data in knowledge graphs, because it allows the identification of each particular descriptive statement, each described entity, quality, and relation, enabling the decomposition of the data graph into various fragments. This characteristic of instance-based ABox semantic graphs is not limited to semantic descriptions of phenotypes, but applies to the description of any type of ODU, including all sorts of particular material entities, spaces, and processes. Thus, when describing a particular entity, instance-based semantic graphs are in general superior to class-based semantic graphs for the same reasons that Phenotype Knowledge Graphs are superior to Semantic Phenotypes. Coupled with their better querying properties, Phenotype Knowledge Graphs together with semantic technologies provide a promising framework for developing not only new innovative analytical methods but also new applications that will substantially support everyday research in the life sciences. We could, for instance, develop algorithms for taxonomists that facilitate statistical evaluation of species affiliation in an anatomically heterogeneous population based on phenotype descriptions. Once we can semi-automatically annotate images and automatically produce Phenotype Knowledge Graphs based on these annotations, the algorithms could compare these graphs and identify putative sub-populations and even suggest adequate diagnostic characters to differentiate these sub-populations. As soon as taxonomists then decide which specimen is the holotype, the algorithms could statistically evaluate the Phenotype Knowledge Graphs of all other previously described specimens belonging to the taxon and generate a consensus description containing all possible conditions found in that taxon. This could be done automatically, and the consensus description would be adjusted dynamically with every new specimen of that taxon being described. Phenotype Knowledge Graphs could even be used for taxonomically identifying the species affiliation of a described specimen. All the resulting information could be documented in a Taxonomy Knowledge Graph, which could provide various valuable services to the life science community, such as automatically generated dynamic multi-entry keys that could add annotated images to each of their decision points. In any case, being able to represent the anatomy of particular specimens in a machine-readable and machine-actionable format is not only going to change the way anatomical research will be done in the future, but it will also increase the visibility and importance of anatomy/morphology and taxonomy as scientific disciplines. Exciting times ahead for morphologists! [fig] Figure 4: Metadata about a Phenotype Knowledge Graph. The Phenotype Knowledge Graph from [/fig] [table] Table 1: Potential suitability of TBox and ABox semantic graphs for meeting eScience-compliant data and metadata standards, using Semantic Phenotypes and Phenotype Knowledge Graphs as examples of data and metadata statements of Phenotype Knowledge Graphs possess their own URI and can be individually referenced.Format standardWhich syntax and file format must be used?Semantic Phenotype✓ Semantic Phenotypes can be documented in RDF/OWL, which provides a machineactionable syntax and format. SPARQL can be used for querying, but querying is computationally more difficult than querying Phenotype Knowledge Graphs.✓✓ Phenotype Knowledge Graphs can be documented in RDF/OWL, which provides a machine-actionable syntax and format. SPARQL can be used for querying. Querying Phenotype Knowledge Graphs is computationally less difficult than querying Semantic Phenotypes.Semantic Phenotype✓ The use of domain-specific semantic data models in Semantic Phenotypes provides a basic categorization and classification of contents relevant for a given domain.✓✓ The use of domain-specific semantic data models in Phenotype Knowledge Graphs provides a basic categorization and classification of contents relevant for a given domain.With the identification of individual descriptive statements, parts, properties, qualities, and relations, Phenotype Knowledge Graphs can be categorized and classified at various levels of granularity, including levels finer than it is possible with Semantic Phenotypes. [/table] [table] Table 2: Potential suitability of TBox and ABox semantic graphs for meeting the FAIR Guiding Principles, using Semantic Phenotypes and Phenotype Knowledge Graphs as examples (criteria taken from[7], criteria for reusability not shown)F1 (meta) data are assigned a globally unique and persistent identifier Semantic Phenotype ✓ Semantic Phenotypes reference ontology classes through their URIs, including the class defining the phenotype. Ontologies provide persistent identifiers for kind terms and their associated universal statements.✓✓ Phenotype Knowledge Graphs not only reference ontology classes like Semantic Phenotypes do, but also provide URIs for every particular descriptive statement, described part, property, quality, and relation and thus for kind terms, universal statements, proper names, and assertional statements.Semantic Phenotype ✓ Metadata can be associated with a phenotype description as a whole, but not with each of the individual descriptive statements it comprises.✓✓ Due to the possibility to organize a Phenotype Knowledge Graph into a set of named graphs, each of which documenting an individual descriptive statement, metadata can be associated on the fine granular level of particular descriptive statements of a phenotype description, in addition to the description as a whole.F3. metadata clearly and explicitly include the identifier of the data it describesSemantic Phenotype ✓ Metadata can include an identifier that refers to the description as a whole, but not to individual descriptive statements.✓✓ Metadata can include an identifier that refers to the description as a whole, but also identifiers that refer to each individual descriptive statement.F4. (meta) data are registered or indexed in a searchable resourceSemantic Phenotype ✓✓ Metadata can be expressed as TBox or ABox semantic graphs and stored in a tuple store.A1.(meta) data are retrievable by their identifier using a standardized communication protocolSemantic Phenotype ✓ Semantic Phenotypes and their metadata can be stored in a tuple store and queried with SPARQL.✓✓ Phenotype Knowledge Graphs and their metadata can be stored in a tuple store and queried with SPARQL. Because particular descriptive statements, described parts, properties, qualities, and relations have their own URIs, they can be individually accessed.A1.1the protocol is open, free, and universally implementable the protocol allows for an authentication and authorization procedure, where necessarySemantic Phenotype -This depends on the application employing the concept of Semantic Phenotypes or Phenotype Knowledge Graphs. A2. metadata are accessible, even when the data are no longer available Semantic Phenotype -This depends on the application employing the concept of Semantic Phenotypes or Phenotype Knowledge Graphs. data use a formal, accessible, shared, and broadly applicable language for knowledge representation Semantic Phenotype ✓ Semantic Phenotypes and Phenotype Knowledge Graphs both can be represented in RDF/OWL. Semantic Phenotype ✓ Semantic Phenotypes and Phenotype Knowledge Graphs both use ontologies and other controlled vocabularies that provide URIs for their terms. [/table] [bib_ref] Signs and terminology: science caught between language and perception, Vogt [/bib_ref] [bib_ref] Minimum information about a microarray experiment (MIAME)-toward standards for microarray data, Brazma [/bib_ref]

The BNT162b2 vaccine induces humoral and cellular immune memory to SARS-CoV-2 Wuhan strain and the Omicron variant in children 5 to 11 years of age SARS-CoV-2 mRNA vaccines prevent severe COVID-19 by generating immune memory, comprising specific antibodies and memory B and T cells. Although children are at low risk of severe COVID-19, the spreading of highly transmissible variants has led to increasing in COVID-19 cases and hospitalizations also in the youngest, but vaccine coverage remains low. Immunogenicity to mRNA vaccines has not been extensively studied in children 5 to 11 years old. In particular, cellular immunity to the wild-type strain (Wuhan) and the cross-reactive response to the Omicron variant of concern has not been investigated. We assessed the humoral and cellular immune response to the SARS-CoV-2 BNT162b2 vaccine in 27 healthy children. We demonstrated that vaccination induced a potent humoral and cellular immune response in all vaccinees. By using spike-specific memory B cells as a measurable imprint of a previous infection, we found that 50% of the children had signs of a past, undiagnosed infection before vaccination. Children with pre-existent immune memory generated significantly increased levels of specific antibodies, and memory T and B cells, directed against not only the wild type virus but also the omicron variant. SARS-CoV-2 mRNA vaccines prevent severe COVID-19 by generating immune memory, comprising specific antibodies and memory B and T cells. Although children are at low risk of severe COVID-19, the spreading of highly transmissible variants has led to increasing in COVID-19 cases and hospitalizations also in the youngest, but vaccine coverage remains low. Immunogenicity to mRNA vaccines has not been extensively studied in children 5 to 11 years old. In particular, cellular immunity to the wild-type strain (Wuhan) and the cross-reactive response to the Omicron variant of concern has not been investigated. We assessed the humoral and cellular immune response to the SARS-CoV-2 BNT162b2 vaccine in 27 healthy children. We demonstrated that vaccination induced a potent humoral and cellular immune response in all vaccinees. By using spike-specific memory B cells as a measurable imprint of a previous infection, we found that 50% of the children had signs of a past, undiagnosed infection before vaccination. Children with pre-existent immune memory generated significantly increased levels of specific antibodies, and memory T and B cells, directed against not only the wild type virus but also the omicron variant. SARS-CoV-2, Omicron, vaccine, children, immune memory, memory B cells, antigenspecific T cells, antibodies Introduction SARS-CoV-2 infection presents with a wide spectrum of clinical manifestations, ranging from mild to severe systemic disease. Most children are either asymptomatic or develop mild manifestations, with rare pulmonary involvement. Recently, however, after the appearance of the high transmissibility of the Omicron Variant of Concern (VOC), the number of infections and hospitalizations has also increased in the 5 to 11 years old age group [bib_ref] SARS-CoV-2 spread and hospitalisations in paediatric patients during the omicron surge, Belay [/bib_ref]. According to the Italian National Institute of Health (ISS) reports, in the last week of August 2022, COVID-19 infections in individuals below 18 years of age accounted for 12% of the recorded cases (3). Although most of the infected children and adolescents are asymptomatic, severe and sometimes life-threatening complications may occur. In particular, the development of the Multisystem Inflammatory Syndrome in Children (MIS-C), a severe post-infectious hyperinflammatory condition, has been reported to present 2-6 weeks after a typically mild or asymptomatic infection [bib_ref] Multisystem inflammatory syndrome in children (MIS-c), Patel [/bib_ref]. Moreover, the persistence of symptoms long after infection, the so-called "long-COVID", should not be underestimated [bib_ref] Preliminary evidence on long COVID in children, Buonsenso [/bib_ref]. Pediatric COVID-19 also poses public health concerns as transmission from children may cause severe disease in adult household members [bib_ref] Household transmission of SARS-CoV-2 from children and adolescents, Chu [/bib_ref]. Furthermore, the COVID-19 pandemic negatively impacts the psychosocial wellbeing of children and their families, particularly those with special education needs and lower socio-economic status [bib_ref] Vulnerability and resilience in children during the COVID-19 pandemic, Tso [/bib_ref]. Vaccines are the only effective large-scale tool to prevent infection and control the pandemic. Over the last two years, several vaccines against SARS-CoV-2 have been rapidly developed. To date, all commercially available vaccines have been shown to be protective against hospitalization and serious illness after completing the immunization schedule [bib_ref] Assessing vaccine effectiveness against severe COVID-19 disease caused by omicron variant. report..., Feikin [/bib_ref]. In the pediatric population, only mRNA vaccines have been approved, first for adolescents aged 12 to 15 years for Pfizer BNT162b2and 12 to 17 years for Moderna mRNA-1273 vaccine [bib_ref] Evaluation of mRNA-1273 SARS-CoV-2 vaccine in adolescents, Ali [/bib_ref] and later for children between 5 and 11 years of age [bib_ref] Evaluation of the BNT162b2 covid-19 vaccine in children 5 to 11 years..., Walter [/bib_ref]. Vaccine use in children 6 months through 4 years of age has also been approved by the FDA (13). In Italy, mRNA vaccines have been introduced in late December 2021 for children aged 5 to 11. The administration of two doses of the Pfizer-BioNTech vaccine in children aged 5-11 years has been proven to be safe, effective, and capable of eliciting a robust humoral immune response to SARS-CoV-2, comparable to that of the adolescents [bib_ref] Evaluation of the BNT162b2 covid-19 vaccine in children 5 to 11 years..., Walter [/bib_ref]. Specific B-and T-cell memory induced by mRNA vaccines has not been investigated in this population. Vaccines protect from infection because they induce immunological memory [bib_ref] A guide to vaccinology: from basic principles to new developments, Pollard [/bib_ref]. Whereas serum antibodies significantly decline a few months after the last SARS-CoV-2 vaccine dose [bib_ref] Highly specific memory b cells generation after the 2nd dose of bnt162b2..., Goel [/bib_ref] , memory B (17) and T (18) cells persist and increase. In case of infection, pre-formed serum antibodies exert the first-line immediate protection, meanwhile memory B cells rapidly expand and differentiate into plasmablasts responsible for the rapid increase of specific antibodies in the serum [bib_ref] A guide to vaccinology: from basic principles to new developments, Pollard [/bib_ref]. Memory cells also migrate to the site of viral entry . Here, memory B cells secrete neutralizing antibodies that prevent viral spreading, and T cells kill virus-infected cells [bib_ref] Persistent b cell memory after SARS-CoV-2 vaccination is functional during breakthrough infections, Terreri [/bib_ref] [bib_ref] SARS-CoV-2 mRNA vaccination elicits a robust and persistent T follicular helper cell..., Mudd [/bib_ref]. The failure of COVID-19 vaccines to prevent infection and contagion has been attributed to their inability of inducing mucosal immune protection [bib_ref] Persistent b cell memory after SARS-CoV-2 vaccination is functional during breakthrough infections, Terreri [/bib_ref] [bib_ref] Dealing with a mucosal viral pandemic: lessons from COVID-19 vaccines, Mouro [/bib_ref] [bib_ref] Anti-spike mucosal IgA protection against SARS-CoV-2 omicron infection, Havervall [/bib_ref] and to the emergence of variants of concern (VOC) (24) with increased transmissibility. However, the third dose of COVID-19 vaccines strongly boosts immunity and, at the population level, reduces severe COVID-19-associated morbidity and mortality, thanks to the increase of antibodies and memory B and T cells able to neutralize not only the original Wuhan strain but also the other variants that appeared over time [bib_ref] Highly specific memory b cells generation after the 2nd dose of bnt162b2..., Goel [/bib_ref] [bib_ref] Naturally enhanced neutralizing breadth against SARS-CoV-2 one year after infection, Wang [/bib_ref] [bib_ref] Efficient recall of omicron-reactive b cell memory after a third dose of..., Goel [/bib_ref] [bib_ref] SARS-CoV-2 vaccination induces immunological T cell memory able to cross-recognize variants from..., Tarke [/bib_ref]. In a recent study conducted on children and adolescents, vaccination reduced the risk of hospitalization for COVID-19 by two-thirds in children 5 to 11 years old during the Omicron period, while most children with critical courses were unvaccinated [bib_ref] Association of prior BNT162b2 COVID-19 vaccination with symptomatic SARS-CoV-2 infection in children..., Price [/bib_ref]. In adolescents, although the effectiveness of two doses of the BNT162b2 vaccine against hospitalization was lower during the Omicron than the Delta wave, vaccination prevented most life-threatening COVID-19 cases in both periods [bib_ref] Association of prior BNT162b2 COVID-19 vaccination with symptomatic SARS-CoV-2 infection in children..., Price [/bib_ref]. Vaccine effectiveness among adolescents increased after a booster dose (29). Recently, different rates of effectiveness of two doses of the BNT162b2 vaccine have been reported in children from Italy and Singapore. The effectiveness against hospitalization was 41.1% in Italy [bib_ref] Effectiveness of BNT162b2 vaccine against SARS-CoV-2 infection and severe COVID-19 in children..., Sacco [/bib_ref] and 82.7% in Singapore (31) during the Omicron period. The discrepancy between these results may be due to different criteria for hospitalization in the two countries. In this study, we describe the humoral and cell-mediated immune response in children, in order to comprehensively evaluate vaccine immunogenicity in this population. A broad understanding of how SARS-CoV-2 vaccination activates the immune system is necessary to find the best predictors of longterm protection and identify individuals that would benefit from additional vaccine doses also in the pediatric age. Moreover, our understanding of the immunological features associated with the main VOCs will be helpful to inform health policies, including boosting and vaccination schedules. # Methods ## Study design and patients We conducted a cross-sectional study on 30 healthy subjects aged 5 to 11 years old enrolled from February to March 2022 at the pediatric Vaccination Center of Policlinico Umberto I (Sapienza University of Rome) where they received Pfizer-BioNTech (BNT162b2) mRNA vaccine immunization. We excluded subjects who already received one or two doses of Pfizer-BioNTech COVID-19 vaccine prior to enrollment, s u b j e c t s d i a g n o s e d w i t h p r i m a r y o r s e c o n d a r y immunodeficiency or with an ongoing infection and, children taking any immunosuppressive drug. The SARS-CoV-2 vaccine was administered as prescribed, in two doses of 10 mg, 21 days apart. Two blood samples were obtained from each participant for serological and cellular immunity assessment at time 0 (T0), before the first dose, and 7-15 days after the second dose (T1). The study protocol was approved by the Ethical Review Committee of Sapienza, University of Rome, Italy (Prot. 0254/ 2022). The study was performed in accordance with the Good Clinical Practice guidelines, the International Conference on Harmonization guidelines, and the most recent version of the Declaration of Helsinki. Parents of the eligible patients were informed on the study, including its safety profile and supply procedures, and signed the informed consents for vaccination and for the immunological study. A structured questionnaire was administered to the parents to investigate whether the children had had positive NPS or experienced COVID-19 before. Demographic (age, gender, and ethnicity) and clinical data were collected to assess the conditions of these subjects [fig_ref] TABLE 1: Demographic and immunological characteristics of the enrolled children [/fig_ref]. ## Cell isolation and cryopreservation Peripheral blood mononuclear cells (PBMCs) were isolated by Ficoll Paque ™ Plus 206 (Amersham PharmaciaBiotech, Amersham, UK) density-gradient centrifugation and immediately frozen and stored in liquid nitrogen until use. ## Detection of sars-cov-2 specific antibodies SARS-CoV-2 specific antibodies were detected on plasma with the DiaSorin Liaison SARS-CoV-2 TrimericS IgG assay (DiaSorin, Saluggia, Italy). The assay was performed on the LIAISON ® XL chemiluminescence analyzer. Anti-Nucleocapsid IgG were measured by Anti-SARS-CoV-2 NCP ELISA assay (Euroimmun, Lübeck, Germany), which uses a modified nucleocapsid protein that only contains diagnostically relevant epitopes. The assay was performed on an automated I-2P analyzer (Euroimmun, Lübeck, Germany). ## Neutralizing antibodies against the wuhan sars-cov-2 strain The Wuhan SARS-CoV-2 strain [GF1] (B1) was incubated at 100 TCID50 (Median Tissue Culture Infectious Dose) with two-fold serial dilutions of plasma samples (1:8 to 1:512) to determine the microneutralization titer (MNT). All plasma samples were first heat-treated for 30 minutes at 56°C to inactivate complement. Virus-plasma mixtures were kept at 37°C for 1 hour in EMEM cell culture medium (Sigma Aldrich, St. Louis, USA) supplemented with 1X penicillin/ streptomycin (Corning, Glendale, USA) and 2% fetal bovine serum (Corning, Glendale, USA) in 96-well plates. After the incubation, 22.000 Vero E6 cells (ATCC ® CRL-1586) were added to each well and cultured at 37°C for 5 days. MNT was calculated as the serum dilution capable of reducing the cytopathic effect to 50%. Positive and negative serum samples and cell culture control were included in each test. Antibody inhibitory activity of the binding between B.1.1.529 BA.1 RBD and ACE-2 Antibodies able to inhibit the binding of ACE-2 to the B.1.1.529 BA.1 RBD were measured using a colorimetric assay kit (BPSBioscience, San Diego, USA). Briefly, 96 well plates were coated with the RBD protein of the B.1.1.529 BA.1 Variant (1ug/ ml). The next day, plasma was added in four 1:3 serial dilutions (1:10 to 1:270). After, we added biotinylated ACE-2 and, after washing, streptavidin-HRP. The plasma dilution capable of reducing the absorbance of the positive control by 50% is reported. ## Detection of sars-cov-2-specific b cells Detection of antigen-specific memory B cells was performed as previously published (16, 17). Briefly, recombinant biotinylated SARS-CoV-2 spike protein (S1+S2; aa16-1211) was purchased from R&D systems (BT10549) and mixed with streptavidin BUV395 or streptavidin PE (BD Bioscience) at 25:1 ratio and 20:1 ratio respectively for 1 hour at 4C. Streptavidin PE-Cy7 (BD Bioscience) was used as a decoy probe to gate out streptavidin-binding B cells. Previously frozen PBMC samples were stained with 100ng of spike-PE, 100ng of spike-BUV395 and 2 ng of streptavidin PE-Cy7 at 4C for 30 min. Following a wash step, a combination of fluorescent antibodies: CD19-BUV737, CD24-BV711, CD27-BV510, CD38-BV421 and IgM-APC was used for surface staining. Spike-specific memory B cells were identified as CD19+CD24+CD27+PeCy7-PE+BUV395+ (Double positive spike;. Samples were acquired on FACS Symphony (BD Bioscience) and analyzed using FlowJo10.7.1 (BD Bioscience). ## Detection of sars-cov-2-specific t-cell response The frequency of spike-specific T cells was assessed by standard IFN gamma ELISpot. PBMC were plated at 3×10 5 cells/well in ELISpot plates (Human IFN-gamma ELISpot plus kit; Mabtech, Nacka Strand, Sweden) and stimulated with a pool of peptides spanning the whole spike protein of the Wuhan SARS-CoV2 strain, or with a pool of peptides spanning the mutated portion of the Omicron spike protein and, as a control, with a pool of peptides spanning the same region of Wuhan strain spike protein (Miltenyi Biotech, Bergisch Gladbach, Germany). Results are expressed as spot-forming cells (SFC)/ 10 6 PBMCs. Cut-off value was set calculating the mean of the background + 2 SD. # Statistical methods Patients' characteristics are summarized in [fig_ref] TABLE 1: Demographic and immunological characteristics of the enrolled children [/fig_ref]. Immunological variables were compared between the different study times. The data were first tested for normality and homoscedasticity using Shapiro Wilk and Levene's tests and since the assumptions were violated, non-parametric tests were used for the analysis. The Wilcoxon matched pair signed-rank test or the two-tailed Mann-Whitney U-test were used. Categorical variables were compared by Chi-Square exact test. A two-sided p value less than 0.05 was considered to be statistically significant. All statistical analyses were done using GraphPad Prism 9.3.1 (GraphPad Software). # Results ## Subject characteristics Of the 30 children enrolled in the study, three were excluded because they had COVID-19 between the first and second dose. Of the 27 studied, 11 children were male and 16 were female, with a mean age of 8.1 years (SD 2.3) [fig_ref] TABLE 1: Demographic and immunological characteristics of the enrolled children [/fig_ref]. Three subjects (3/27; 11.1%) reported a history of SARS-CoV-2 infection before vaccination (positive molecular Nasopharyngeal Swab, NPS), one 5 months before vaccination and the other two more than a year before. Based on the epidemiology of SARS-CoV-2 in Italy, none of the three children contracted the disease during the Omicron wave. Symptoms were mild (fever, cough, cold, vomit) and resolved in a few days. The infection was detected at the same time in their families. The other 24 children reported no history of infection [fig_ref] TABLE 1: Demographic and immunological characteristics of the enrolled children [/fig_ref]. ## Humoral and cellular sars-cov-2 response after vaccination Anti-TrimericS specific IgG increased significantly after the second dose (T1) compared to T0 (p <0.0001) in all children. In order to evaluate the quality of the antibody response, we measured the neutralization activity against the Wuhan SARS-CoV-2 strain . The increase was significant in all vaccinated children (p<0.001). By flow-cytometry, we measured the frequency of memory B cells able to bind the Wuhan spike protein (Supplementary. Specific memory B cells significantly expanded (p <0.0001) after the second dose. Spike-specific T cells able to produce IFN-gamma after stimulation with peptides spanning the whole Wuhan spike protein (Complete Wuhan spike) were detected by ELISpot. Following vaccination, there was a significant increase of IFNgamma-secreting T cells (p<0.0001) . As shown in , at T0, anti-TrimericS were detectable in 16 children and neutralizing antibodies in 12. Spike-specific memory B and T cells were also measurable in some of the samples collected before vaccination. Since, based on their medical history, only three of the 27 children had experienced COVID-19 before (indicated by the dots with thick borders), we asked the question of whether the presence of pre-existing humoral and cellular immunity might be due to previous undiagnosed SARS-CoV-2 infections. Whereas serum antibodies may have been generated in response to other coronaviruses and cross-reactive T cells can be found in individuals never exposed to SARS-CoV-2 (32), spike-specific memory B cells are only detected in individuals who had experienced COVID-19 before or were fully vaccinated (16, [bib_ref] Sequential analysis of binding and neutralizing antibody in COVID-19 convalescent patients at..., Goel [/bib_ref]. Indeed, the presence of specific memory B cells represent a reliable imprint of past contact with a defined antigen because their generation is a complex process occurring in the germinal c enter (1 7). In these vacc ine-induced structures, immunoglobulin genes are modified by the introduction of somatic mutations, followed by a strict selection for the ability to bind the stimulating antigen. Spike-specific memory B cells persist and continue to increase for months after vaccination [bib_ref] Persistent b cell memory after SARS-CoV-2 vaccination is functional during breakthrough infections, Terreri [/bib_ref] or infection (34), in contrast to specific anti-S and anti-N antibodies that decline over time (35). Based on these observations, we re-analyzed the data by comparing children who had (group 1) or not (group 2) spikespecific memory B cells at T0 [fig_ref] TABLE 1: Demographic and immunological characteristics of the enrolled children [/fig_ref]. Fifteen (55.5%) of the children in our cohort had spikespecific memory B cells at T0 . As only three of them had a documented history of infection, 50% (12/24) of the remaining children had probably experienced an infection that was never detected, although NPS was performed in the subjects who had contact with infected individuals or presented respiratory symptoms. Anti-N antibodies are considered a reliable indicator of a previous SARS-CoV-2 infection. We found that anti-N antibodies were, however, present only in 73% of the children Design of the study. . Anti-N antibodies were undetectable in 27% of group 1, including two children who had experienced COVID-19 more than one year before. The progressive decline of anti-N IgG was confirmed by their reduction at T1, about 30 days after the T0 measurement [fig_ref] TABLE 1: Demographic and immunological characteristics of the enrolled children [/fig_ref] and Supplementary . Thus, due to their continuous decline, anti-N antibodies may fail to identify children who had been infected a long time before the serological test. We found that spike-specific memory B cells increased in both groups after vaccination, but the response was significantly greater in group 1 than in group 2 (p=0.0002). Children of group 1 also had higher levels of anti-TrimericS IgG ; p=0.001) and neutralizing antibodies ; p=0.04) after the 2nd dose. It has been demonstrated that repeated exposures to the SARS-CoV-2 spike protein, either because of infection before or after vaccination or administration of a booster vaccine dose, increase immunity against not only the Wuhan strain but also the Omicron VOC [bib_ref] Three exposures to the spike protein of SARS-CoV-2 by either infection or..., Wratil [/bib_ref]. In order to have a measure of the potential neutralization ability of vaccine-induced antibodies against the Omicron VOC, we measured their ability to inhibit the binding of ACE-2 to the Omicron RBD. The inhibitory activity was increased by vaccination in all children, but group 1 had higher inhibitory antibody titers before (p=0.006) and after vaccination (p=0.01) compared to group 2 . Thus, although after vaccination all children had high titers of anti-TrimericS and neutralizing antibodies against the Wuhan strain, antibodies with inhibitory activity against the Omicron VOC were present in 87% (13/15) of the children in group 1 and only in 42% of those of group 2 (5/12) (p=0.01; . Spike-specific T cells directed against the Wuhan strain spike (complete Wuhan spike) were equally induced in group 1 and group 2 children , and the response rate after vaccination was 100% in both groups . We also measured the frequency of T cells able to recognize the region mutated in the Omicron spike and, as control, its unmutated counterpart (Wuhan reference). We found that, after vaccination, children of group 1 had significantly better T-cell responses than those of group 2 (Omicron spike p=0.04; Wuhan reference p=0.01) . Most importantly, only 18% (2/11) of the children in group 2 had T cells able to recognize the Omicron spike, whereas, in group 1, 71.4% (10/14) of the children were responders . The Wuhan reference of the Omicron spike was recognized by 80% of the children in group 1 and 40% of those in group 2 . Thus, in response to vaccination, children who had spikespecific memory B cells at T0 produced more antibodies, had more memory B cells and antigen-specific T cells, and were able to react against the Omicron VOC, probably as a result of highly effective hybrid immunity [bib_ref] SARS-CoV-2 hybrid immunity: silver bullet or silver lining? nat rev, Suryawanshi [/bib_ref]. For children completely naïve to the virus, two vaccine doses may be insufficient to obtain the same degree of immunity. # Discussion Since its first description in 2019, SARS-CoV-2, the causative agent of COVID-19, continues to accumulate mutations and generate variants. Although children present an asymptomatic or paucisymptomatic course, complications from COVID-19 may occur and pediatric infection represents a public health problem. The highly transmissible Omicron VOC is now dominant all over the world. Although several studies have Humoral and cellular immunity in groups 1 and 2. (A) Pie chart represents the percentage of children with (group 1, n=15) and without (group 2, n=12) spike-specific memory B cells before vaccination (T0). (B) Dot chart depicts the frequency of spike-specific memory B cells in the two groups before (T0) and 10 days after vaccination (T1). Concentration of anti-TrimericS IgG (BAU/mL) and neutralization titers, reported as IC50 against the Wuhan viral strain are shown in (C, D), respectively. (E) Graph indicates the inhibitory activity against the Omicron VOC in the two groups. (F) Bar charts represent the percentage of children in group 1 and group 2 with anti-TrimericS IgG (BAU/mL), neutralizing antibodies against the complete Wuhan spike and antibodies able to inhibit the binding of ACE-2 to the Omicron RBD. Dot charts show the number of T cells, expressed as numbers of spot-forming cells (SFC)/10 6 PBMC, producing IFN gamma after stimulation with peptides encompassing the complete Wuhan spike protein (complete Wuhan spike) (G), the spike region mutated in the Omicron VOC (H) and its unmutated counterpart (I). (J) Bar charts represent the percentage of responders in group 1 and group 2 against the complete Wuhan spike protein, the spike region mutated in the Omicron VOC and its unmutated counterpart. Dashed lines indicate the cutoff value for each test. Bars indicate medians and dots with thick borders show values measured in children that had experienced COVID-19 before vaccination (n=3). Non-parametric Wilcoxon matched pair signed-rank test (continuous line) and Mann-Whitney t-test (dashed line) were used to evaluate statistical significance. Categorical variables were compared by Chi-Square exact test. Two-tailed P value significances are shown as * p<0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. shown that the Omicron wave is associated with milder illness and an overall lower hospitalization rate [bib_ref] Decreased severity of disease during the first global omicron variant covid-19 outbreak..., Abdullah [/bib_ref] , the number of pediatric patients affected by Omicron exceeds the total number of cases seen in previous waves [bib_ref] Coronavirus disease 2019 transmission and symptoms in young children during the severe..., Akaishi [/bib_ref] , ultimately leading to an increase in the absolute number of pediatric patients with hospitalization and severe outcomes [bib_ref] SARS-CoV-2 spread and hospitalisations in paediatric patients during the omicron surge, Belay [/bib_ref]. Vaccination is the best strategy to reduce the severity of the disease and limit complications of COVID-19. It has been shown that BNT162b2 vaccination can consistently reduce Omicron-associated hospitalizations in children [bib_ref] Association of prior BNT162b2 COVID-19 vaccination with symptomatic SARS-CoV-2 infection in children..., Price [/bib_ref] [bib_ref] Risk of infection and hospitalization among vaccinated and unvaccinated children and adolescents..., Dorabawila [/bib_ref] and prevent or reduce the associated complications, such as MIS-C (42), long COVID (43), and impairment of social and mental wellbeing [bib_ref] Manifesto of the pediatricians of Emilia-romagna region, Italy, in favor of vaccination..., Esposito [/bib_ref]. After being recommended for adolescents, the BNT162b2 mRNA vaccine was approved and found to be safe, immunogenic, and efficacious in children 5 to 11 years old [bib_ref] Evaluation of the BNT162b2 covid-19 vaccine in children 5 to 11 years..., Walter [/bib_ref] [bib_ref] Short-term side effects and SARS-CoV-2 infection after COVID-19 pfizer-BioNTech vaccine in children..., Capponi [/bib_ref] , and, most recently, in children with an age between 4 months and 4 years (13). The immune response to pediatric vaccination has not yet been fully elucidated, and specifically, an understanding of the role of cellular immunity to infection and vaccination is missing at this age [bib_ref] mRNA-based COVID-19 vaccine boosters induce neutralizing immunity against SARS-CoV-2 omicron variant, Healy [/bib_ref]. Indeed, the registration studies have focused on neutralizing antibody levels for immunobridging [bib_ref] Evaluation of the BNT162b2 covid-19 vaccine in children 5 to 11 years..., Walter [/bib_ref]. Although antibodies are a reliable measure of vaccine efficacy, memory B and T cells are important for long-term protection and are capable of responding to emerging VOC [bib_ref] Highly specific memory b cells generation after the 2nd dose of bnt162b2..., Goel [/bib_ref]. We studied the humoral and cellular immune response to the BNT162b2 vaccine encoding the spike protein of the Wuhan viral strain. All children responded to the vaccine with a significant increase of anti-TrimericS IgG, neutralizing antibodies, spike-specific memory B cells and antigen-specific T cells . We have previously published that, in adults (16, 17), spikespecific memory B cells are undetectable before vaccination and develop after a complete cycle, because they are generated by the complex mechanism that modifies immunoglobulin genes and increases antibody affinity. Unexpectedly, we detected circulating spike-specific memory B cells before administration of the first dose in 15 of 27 children . Only three of them had a documented history of COVID-19, suggesting that in the other 12 individuals, memory B cells may have been generated by undetected and asymptomatic SARS-CoV-2 infections. All infections known (3 cases) and undiagnosed (12 cases) occurred before the Omicron variant spread in Italy. This hypothesis was confirmed by the presence of anti-TrimericS IgG, neutralizing antibodies, and Spike-specific T cells in the samples collected at T0 . Anti-N IgG were detectable in the majority, but not all the children who had pre-existing immunity to SARS-CoV-2, probably because of the anti-N antibodies physiological decline, months after the infection (35) . In comparison to children without pre-existing antigen-specific memory B cells, those with established B and T cells immunity had a response to the vaccine that was not only significantly stronger but also broader, as demonstrated by the presence of antibodies with inhibitory activity against the Omicron VOC and T cells specific for the mutated regions of the Omicron Spike . Our data demonstrate that the presence of spike-specific memory B cells identifies individuals who had a previous undiagnosed encounter with the virus. Based on the information obtained from the structured questionnaire that was administered to the parents to investigate whether the children had had positive NPS or experienced COVID-19 before, 80% (12/15) of the children with pre-existing immunity had experienced an asymptomatic and undiagnosed infection. This infection had, however, generated persistent memory B and T cells, responsible for the strong reaction to the vaccine, typical of hybrid immunity. As a broader response, anti-Omicron neutralizing antibodies are induced after a booster dose in adults and adolescents (47, 48), our results suggest that a third vaccine dose may amplify the amount and breadth of vaccine-induced immunity also in children. A primary response to immunization is directed against the more immunogenic dominant epitopes [bib_ref] Masking of antigenic epitopes by antibodies shapes the humoral immune response to..., Zarnitsyna [/bib_ref]. Repeated exposures to the spike protein, because of booster doses or natural infection, may redirect the response to other regions of the immunizing antigen and thus explain the increase of Omicron neutralizing antibodies (50) and of T cells recognizing the Spike regions mutated in the Omicron VOC (51) . The CDC has approved the administration of a homologous booster dose for children aged 5-11 years and reviewed the data demonstrating the safety of the procedure (52). Most recently, bivalent COVID-19 booster doses have been authorized in the USA also for children ages 5 and above, with the aim of broadening and strengthening the protection against the currently dominant VOC. Furthermore, a recent study indicates that protection against Omicron infection generated by two mRNA vaccine doses or by infection rapidly declines, suggesting that children may benefit from a booster dose of vaccine [bib_ref] Effects of vaccination and previous infection on omicron infections in children, Lin [/bib_ref]. In Italy, the third dose, either with the homologous or bivalent vaccine, has not yet been approved for children of this age. The SARS-CoV-2 pandemic has been caused by the lack of pre-existing immunity to a virus never encountered before by humans. Vaccines have reduced the number of severe cases and deaths thanks to the generation of immune memory. Although so far, all vaccinated people are all immunized against the Wuhan Spike protein, vaccine-induced memory B and T cells are also able to react to the Omicron variant. The major strength of our study is the comprehensive evaluation of the immune response to COVID-19 vaccination in 5 to 11 years old children. Until now, all published studies have measured serum antibodies. Here, we also demonstrate that B and T cell memory is elicited and that repeated antigen encounters lead to a broader response directed against the Wuhan virus and also the Omicron VOC. We also show that spike-specific memory B cells are a reliable indicator of a previous infection. Our work has major limitations. We analyzed a limited number of subjects due to the difficulty of recruiting children in a study that requires repeated blood sample collections. We did not evaluate the neutralization activity of vaccine-induced antibodies against Omicron with the classic neutralization test, because the system was not available in our laboratories. We, however, measured the ability of vaccine-induced antibodies to block the binding of ACE-2 to the Omicron RBD, thus measuring specificity and activity against Omicron. Evaluation of memory B cells specific for Omicron was impossible due to the small amount of blood available for our study. In conclusion, we provide the demonstration that the BNT162b2 vaccine is immunogenic in children and repeated antigen exposures may increase the ability to react toward the emerging VOC. The results of this study offer new insights into the humoral and cellular response to SARS-CoV-2 mRNA vaccines in children, useful to support public health decisions on the definition of future vaccination strategies in the pediatric age. Improving knowledge on immune response following vaccination may also help overcome vaccine hesitancy, considering that children, as adults, have the right to be protected from severe disease and COVID-19 complications and sequelae [bib_ref] Manifesto of the pediatricians of Emilia-romagna region, Italy, in favor of vaccination..., Esposito [/bib_ref]. # Data availability statement The raw data supporting the conclusions of this article will be made available by the authors, without undue reservation. # Ethics statement The studies involving human participants were reviewed and approved by Ethical Review Committee of Sapienza, University of Rome, Italy (Prot. 0254/2022). Written informed consent to participate in this study was provided by the participants' legal guardian/next of kin. # Author contributions BLC, EPM, VB, ChrA, CA, GF, IS, PS, SF, and MC acquired the data. BLC, EPM and RC were responsible for the analysis of the data. BLC, EPM and RC drafted the article. AMZ, CA, MGC, LP, ATP, PS, AS, FM, IS, FL critically revised the manuscript for important intellectual content. All authors approved the final version to be published and agree to be held accountable for all aspects of the work. BLC, EPM and RC had full access to, and verified, all the data in the study, and had final responsibility for the decision to submit for publication. RC, CA and FL made substantial contributions to the acquisition of funding. All authors contributed to the article and approved the submitted version. # Funding The study was supported by funding of the Italian Ministry of Health COVID2020-12371817 and by Grant ''5 per mille, 2021''. [table] TABLE 1: Demographic and immunological characteristics of the enrolled children. [/table]

The cellular environment shapes the nuclear pore complex architecture Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE) 1-3 . These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel 4-6 . Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture.A combination of X-ray crystallography and electron microscopy (EM) approaches has generated a consensus model for the NPC architecture, characterized by three substructures that span the NE 4,5,7 . The inner ring (IR) is centrally positioned at the fusion point of the inner and outer nuclear membranes (INM and ONM, respectively) and forms the central channel that connects to phenylalanine-glycine (FG) repeat proteins that create the permeability barrier and facilitate active cargo transport. The cytoplasmic ring (CR) provides a platform for protein and RNA export 3,8 . The nucleoplasmic ring (NR) shares scaffold nucleoporins (Nups) with the CR and anchors a distinct basket-like structure with diverse functions.Although all eukaryotes maintain a three-ring NPC structure, the composition and arrangement of the substructures varies between species. In mammalian cells, the IR has five primary subunits: Nup205, Nup188, Nup155, Nup93 and Nup35 (refs. 4,5,9 ). FG-Nups (Nup54, Nup58 and Nup62) are docked to the IR to create the central channel for transport 4,5,7 . The CR and NR both contain 16 copies of the Y complex 10-13 , which contains a set of elongated helical stack proteins (Nup160, Nup133, Nup107, Nup96 and Nup85) decorated with the beta-propellers Sec13, Seh1, Nup37 and Nup43 (refs. 11,12,14 ). The principal Y-complex arrangement in both the CR and NR follows a reticulated pattern composed of two eight-membered rings 12,15 .Our current understanding of the human NPC structure is based mainly on cryo-electron tomography (cryo-ET) studies of NPCs from purified NEs at 2-6 nm resolution 12,15,16 . Cryo-focused-ion-beam (cryo-FIB) milling now enables the study of macromolecular complexes in their native environment 17 . NPCs from cryo-FIB-milled yeast 18,19 , algae 20 and human cells21,22show differences compared with previous human NPC structures, including wider IRs. Here we study the architecture of the human NPC from cryo-FIB-milled DLD-1 cells. Our data establish concepts in human NPC architecture that may have previously been interpreted as species-specific differences. Our study shows that the cellular environment substantially influences the diameter of the NPC central channel and emphasizes the modular, although interdependent, architecture of the NPC and its role in shaping the NE.The architecture of the NPC in DLD-1 cellsWe used cryo-FIB-milled lamellae of human DLD-1 cells containing an auxin-inducible degron (AID) tag at the NUP96 (HGNC symbol: NUP98) loci (homozygous NUP96::Neon-AID) 23 for the targeted depletion of Nup96. Such Nup96 depletion leads to a loss of both the CR and NR elements, whereas the IR is unaffected. Here we compared native and partially degraded NPC structures. Lamellae were prepared from cells in the absence of auxin (wild-type condition) for cryo-ET imaging and identification of in situ NPCs. We extracted 194 NPC-containing subvolumes from 54 tomograms and used subtomogram averaging to obtain a density map of the NPC resolved to ~3.4 nm (Fig. 1and Extended DataFig. 1a-c). Our structural analysis revealed a similar three-ring architecture to that observed in previous human models12,15,16. However, the diameter of Article the central channel is considerably different(Fig. 1cand Extended DataFig. 2a)-it is about 33% larger (width, about 57 nm) compared with previous models (width, about 43 nm) 15 . Furthermore, the outer diameter of the IR is about 105 nm, compared with around 89 nm for semi-purified NPCs. Moreover, we observed a reduced NPC height of 66 nm, compared with 75 nm in previous models. Finally, the distance between the centre of the IR and the distal end of the CR is greater than the distance between the IR and the NR (37 nm versus 29 nm, respectively)(Fig. 1c).We fit structures of nucleoporin subcomplexes into the three rings of the averaged map (Fig. 1cand Extended DataFig. 1d-f). For the NR and CR, we used a composite Y-complex model derived from overlapping crystal structures and threaded models11,14,15,24. Bending at recognized 'hinge points' 10,11,14 , we fit the Y complex (Extended DataFig. 3a-c). It docks into both the CR and NR in a reticulated pattern of eight-membered rings (Extended DataFig. 4a-e). In each ring, we observed considerable density beyond the Y complexes. The additional density in the CR might correspond to Nup358, an expected component of this subassembly 15 (Extended DataFig. 4b). We also observed density protruding out of the CR towards the centre of the ring at the base of Nup85 (Extended DataFig. 1d). On the basis of previous cross-linking8,25and cryo-EM/cryo-ET 26,27 studies, this density is probably occupied by the Nup214-Nup88-Nup62 complex.For the IR, we first docked the published composite model containing Nup93, Nup188/Nup205, Nup155 and the Nup62-Nup58-Nup54 complex 9 (Extended DataFig. 3d, e). Although this model fits well, some elements appear to be shifted, suggesting that flexibility in the IR Nups may account for the larger diameter that we observed (Extended DataFig. 5a).To better fit the model, we isolated individual Nups and reoriented them locally to create an updated IR model (ExtendedData Fig. 5a, b).The NR is also composed of two eight-membered rings of Y complexes. As in the CR, we identified additional density near the Nup85 arms (Extended DataFig. 1f), and observed a linker density between the Y rings in agreement with a previous report 15 (Extended DataFig. 4c). Unique to the NR, we observed density at the base of the Y-complex-containing rings, which extends towards the IR (Extended DataFig. 5e, f).Asymmetric nuclear membranes at the NPCCross-sections through the cryo-ET map reveal that the NE at the NPC is asymmetric, with a steeper angle at the CR compared with at the NR (about 42° compared with about 28°, respectively), using the IR as a plane of reference(Fig. 2a). This membrane asymmetry is also evident in two-dimensional (2D) orthoslices of eight-fold symmetrized NPCs. In different planes of the NPC, it is readily apparent how the NE is shallower at the nucleoplasmic versus the cytoplasmic surface(Fig. 2b).Native NPC has a larger central channelCompared with previous human NPC models, our map displays substantial differences, including larger IR and central channel diameters. Although the IR is essential for maintaining the architecture of the NPC central channel, it also anchors Nups with flexible, unstructured FG-rich domains that facilitate transport through the channel 7,28 . We found that the channel delineated by the IR has a volume that is about 75% larger than previously reported 15(Fig. 3a). We expect that these differences are due to the preserved cellular environment rather than any cell-type-specific differences, as we observed a similar architecture in HeLa-derived cells (Extended DataFig. 2a, b). Furthermore, we analysed data from mouse embryonic fibroblasts in which cryo-ETs of NPCs were captured by cryo-FIB milling or cell permeabilization followed by nuclease incubation 29 . The diameter of NPCs from cryo-FIB cells was again notably larger compared with the diameter of NPCs from permeabilized cells (ExtendedData Fig. 2c). a b CR NR IR 105 nm 37 nm 29 nm 57 nm NR CR 180°N ucleus Cytoplasm CR CR NR NR IR IR c Fig. 1 | The NPC structure from human DLD-1 cells. a, Tomographic slice from a lamella of a NUP96::Neon-AID DLD-1 cell in the absence of auxin (wild-type condition). The slice thickness is 2.7 nm. The arrowheads indicate each of the NPC rings. The arrow indicates an adjacent NPC. Scale bar, 100 nm. b, Whole NPC map from DLD-1 cells. Scale bar, 20 nm. c, Cross-section of a cryo-ET map with fitted structures for the CR (blue and yellow), IR (orange and magenta) and NR (light blue and gold). The central channel diameter (57 nm), the membrane-to-membrane diameter (105 nm) and the height across the NE (37 nm from IR-CR, 29 nm from IR-NR) are indicated. CR NR IR 2 3 1 2 3 90°1 2 3 1 2 3 CR Y attachment NR Y attachment No attachments CR NR IR 28°4 2°1 a b Fig. 2 | Asymmetric nuclear membrane at the NPC. a, Cross-section of the NPC structure. The angles indicate measurements from the membrane distal edge of the IR to the edge of the nuclear membrane at the NR or CR. b, 2D orthoslices through the full-NPC average. Top, NPC map; the planes of the 2D slices (bottom) are indicated. Scale bar, 20 nm. Plane 1, at the point of the interaction between the CR Y complex and the membrane. Plane 2, at the point of the interaction between the NR Y complex and the membrane. Plane 3: sSlice in which no Y complexes touch membrane. The slice thickness is 1.4 nm. Nature | Vol 598 | 28 October 2021 | 669In our IR model, Nup205/Nup188 (paralogues that are indistinguishable at this resolution), Nup155 and Nup93 are significantly shifted, whereas the Nup62-Nup58-Nup54 complex is stable (Extended DataFig. 5b). Propagated through the entire IR, these rearrangements explain how the central channel becomes larger. Adjacent IR subunits are nearly separated compared with previous models in which the subunits were snugly aligned(Fig. 3a, band Extended DataFig. 5c, d). The Nup93 and Nup205 subunits of adjacent IR structures form a pseudo-lock-and-key configuration in the constricted IR model; by contrast, here these components shifted to create gaps between the IR subunits(Fig. 3c).In contrast to the IR, the diameters of the CR and NR are nearly identical to previous models (Extended DataFig. 4d, e). However, to accommodate a flatter overall assembly, the CR and NR rings flex in relation to the NE. At both the CR and NR, the Y-complex rings are angled more inward towards the central channel (Extended DataFig. 4f).Architecture of Nup96-depleted NPCsTo better understand the contribution of each ring to the overall NPC assembly, we next analysed the structure of Nup96-depleted NPCs. We acquired 71 tomograms of cryo-FIB-milled NUP96::Neon-AID DLD-1 cells after auxin-induced Nup96 depletion and extracted 163 NPCcontaining subvolumes for subtomogram averaging (Extended DataFig. 6a-f). In two cells, we observed ONM herniations (Extended DataFig. 6g), indicating dysfunctional assembly of NPCs 30 . NPCs from this dataset could be divided into the following three classes(Fig. 4a): class 1, with all three rings (17% of particles); class 2, with NR-IR complexes (32%); and class 3, containing only the IR (51%). As we found no CR-IR complexes, this suggests that the CR is more readily removed from the NPC than the NR after Nup96 depletion. Furthermore, the diameters of each unique NPC assembly varied, and assemblies with NR-IR or IR only had significantly larger diameters (Extended DataFig. 6c). With the reduction from three to two to one ring, the spacing between the eight IR subcomplexes increases(Fig. 4a). In addition to the dilated central channel in IR-only NPCs, a cross-section analysis revealed changes in membrane orientations at the opening surrounding Nup96-depleted NPCs(Fig. 4a). The membrane has a smaller radius of curvature at the ONM-INM fusion, and the NE is more symmetric compared with in wild-type NPCs.DiscussionThe architectural analysis of the NPC is a major challenge for structural biology. To date, the most accurate models of the human 57 nm 43 nm 105 nm 89 nm 90°90°S ubunit i i -1 i +1 Subunit i i -1 i +1 DLD-1 cryo-FIB This study HeLa isolated NEs EMD-3103 IR channel Volume: 51 × 10 3 nm 3 IR channel Volume: 29 × 10 3 nm 3 a b c Fig. 3 | IR flexibility increases central channel size in native NPCs. a, Segmentation of the IR from our model and the previous human NPC model. The IR diameter (membrane to membrane) and central channel diameter measurements are indicated. The IR channel volume was calculated as a cylinder with a height of 20 nm. b, Top view of three IR subunits in our model compared with the previous model. c, Schematic showing the lock-and-key-like architecture of the previous model and how the IR subunits have shifted in our model. Nuclear pore complexes (NPCs) create large conduits for cargo transport between the nucleus and cytoplasm across the nuclear envelope (NE) [bib_ref] Functional architecture of the nuclear pore complex, Grossman [/bib_ref] [bib_ref] The nuclear pore complex as a flexible and dynamic gate, Knockenhauer [/bib_ref] [bib_ref] The nuclear pore complex: bridging nuclear transport and gene regulation, Strambio-De-Castillia [/bib_ref]. These multi-megadalton structures are composed of about thirty different nucleoporins that are distributed in three main substructures (the inner, cytoplasmic and nucleoplasmic rings) around the central transport channel [bib_ref] Structure and assembly of the nuclear pore complex, Hampoelz [/bib_ref] [bib_ref] The structure of the nuclear pore complex (an update), Lin [/bib_ref] [bib_ref] The structure inventory of the nuclear pore complex, Schwartz [/bib_ref]. Here we use cryo-electron tomography on DLD-1 cells that were prepared using cryo-focused-ion-beam milling to generate a structural model for the human NPC in its native environment. We show that-compared with previous human NPC models obtained from purified NEs-the inner ring in our model is substantially wider; the volume of the central channel is increased by 75% and the nucleoplasmic and cytoplasmic rings are reorganized. Moreover, the NPC membrane exhibits asymmetry around the inner-ring complex. Using targeted degradation of Nup96, a scaffold nucleoporin of the cytoplasmic and nucleoplasmic rings, we observe the interdependence of each ring in modulating the central channel and maintaining membrane asymmetry. Our findings highlight the inherent flexibility of the NPC and suggest that the cellular environment has a considerable influence on NPC dimensions and architecture. A combination of X-ray crystallography and electron microscopy (EM) approaches has generated a consensus model for the NPC architecture, characterized by three substructures that span the NE [bib_ref] Structure and assembly of the nuclear pore complex, Hampoelz [/bib_ref] [bib_ref] The structure of the nuclear pore complex (an update), Lin [/bib_ref] [bib_ref] One ring to rule them all? Structural and functional diversity in the..., Fernandez-Martinez [/bib_ref]. The inner ring (IR) is centrally positioned at the fusion point of the inner and outer nuclear membranes (INM and ONM, respectively) and forms the central channel that connects to phenylalanine-glycine (FG) repeat proteins that create the permeability barrier and facilitate active cargo transport. The cytoplasmic ring (CR) provides a platform for protein and RNA export [bib_ref] The nuclear pore complex: bridging nuclear transport and gene regulation, Strambio-De-Castillia [/bib_ref] [bib_ref] Structure and function of the nuclear pore complex cytoplasmic mRNA export platform, Fernandez-Martinez [/bib_ref]. The nucleoplasmic ring (NR) shares scaffold nucleoporins (Nups) with the CR and anchors a distinct basket-like structure with diverse functions. Although all eukaryotes maintain a three-ring NPC structure, the composition and arrangement of the substructures varies between species. In mammalian cells, the IR has five primary subunits: Nup205, Nup188, Nup155, Nup93 and Nup35 (refs. [bib_ref] Structure and assembly of the nuclear pore complex, Hampoelz [/bib_ref] [bib_ref] The structure of the nuclear pore complex (an update), Lin [/bib_ref] [bib_ref] Molecular architecture of the inner ring scaffold of the human nuclear pore..., Kosinski [/bib_ref]. FG-Nups (Nup54, Nup58 and Nup62) are docked to the IR to create the central channel for transport [bib_ref] Structure and assembly of the nuclear pore complex, Hampoelz [/bib_ref] [bib_ref] The structure of the nuclear pore complex (an update), Lin [/bib_ref] [bib_ref] One ring to rule them all? Structural and functional diversity in the..., Fernandez-Martinez [/bib_ref]. The CR and NR both contain 16 copies of the Y complex [bib_ref] Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore..., Kampmann [/bib_ref] [bib_ref] Atomic structure of the Y complex of the nuclear pore, Kelley [/bib_ref] [bib_ref] Integrated structural analysis of the human nuclear pore complex scaffold, Bui [/bib_ref] [bib_ref] Modular self-assembly of a Y-shaped multiprotein complex from seven nucleoporins, Lutzmann [/bib_ref] , which contains a set of elongated helical stack proteins (Nup160, Nup133, Nup107, Nup96 and Nup85) decorated with the beta-propellers Sec13, Seh1, Nup37 and Nup43 (refs. [bib_ref] Atomic structure of the Y complex of the nuclear pore, Kelley [/bib_ref] [bib_ref] Integrated structural analysis of the human nuclear pore complex scaffold, Bui [/bib_ref] [bib_ref] Architecture of the nuclear pore complex coat, Stuwe [/bib_ref]. The principal Y-complex arrangement in both the CR and NR follows a reticulated pattern composed of two eight-membered rings [bib_ref] Integrated structural analysis of the human nuclear pore complex scaffold, Bui [/bib_ref] [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref]. Our current understanding of the human NPC structure is based mainly on cryo-electron tomography (cryo-ET) studies of NPCs from purified NEs at 2-6 nm resolution [bib_ref] Integrated structural analysis of the human nuclear pore complex scaffold, Bui [/bib_ref] [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] [bib_ref] The human nuclear pore complex as revealed by cryo-electron tomography, Maimon [/bib_ref]. Cryo-focused-ion-beam (cryo-FIB) milling now enables the study of macromolecular complexes in their native environment [bib_ref] Opening windows into the cell: focused-ion-beam milling for cryo-electron tomography, Villa [/bib_ref]. NPCs from cryo-FIB-milled yeast [bib_ref] In-cell architecture of the nuclear pore and snapshots of its turnover, Allegretti [/bib_ref] , algaeand human cells [bib_ref] Visualizing the molecular sociology at the HeLa cell nuclear periphery, Mahamid [/bib_ref] [bib_ref] Cone-shaped HIV-1 capsids are transported through intact nuclear pores, Zila [/bib_ref] show differences compared with previous human NPC structures, including wider IRs. Here we study the architecture of the human NPC from cryo-FIB-milled DLD-1 cells. Our data establish concepts in human NPC architecture that may have previously been interpreted as species-specific differences. Our study shows that the cellular environment substantially influences the diameter of the NPC central channel and emphasizes the modular, although interdependent, architecture of the NPC and its role in shaping the NE. ## The architecture of the npc in dld-1 cells We used cryo-FIB-milled lamellae of human DLD-1 cells containing an auxin-inducible degron (AID) tag at the NUP96 (HGNC symbol: NUP98) loci (homozygous NUP96::Neon-AID)for the targeted depletion of Nup96. Such Nup96 depletion leads to a loss of both the CR and NR elements, whereas the IR is unaffected. Here we compared native and partially degraded NPC structures. Lamellae were prepared from cells in the absence of auxin (wild-type condition) for cryo-ET imaging and identification of in situ NPCs. We extracted 194 NPC-containing subvolumes from 54 tomograms and used subtomogram averaging to obtain a density map of the NPC resolved to ~3.4 nm and Extended Data . Our structural analysis revealed a similar three-ring architecture to that observed in previous human models [bib_ref] Integrated structural analysis of the human nuclear pore complex scaffold, Bui [/bib_ref] [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] [bib_ref] The human nuclear pore complex as revealed by cryo-electron tomography, Maimon [/bib_ref]. However, the diameter of Article the central channel is considerably different and Extended Data -it is about 33% larger (width, about 57 nm) compared with previous models (width, about 43 nm) [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref]. Furthermore, the outer diameter of the IR is about 105 nm, compared with around 89 nm for semi-purified NPCs. Moreover, we observed a reduced NPC height of 66 nm, compared with 75 nm in previous models. Finally, the distance between the centre of the IR and the distal end of the CR is greater than the distance between the IR and the NR (37 nm versus 29 nm, respectively) . We fit structures of nucleoporin subcomplexes into the three rings of the averaged map and Extended Data . For the NR and CR, we used a composite Y-complex model derived from overlapping crystal structures and threaded models [bib_ref] Atomic structure of the Y complex of the nuclear pore, Kelley [/bib_ref] [bib_ref] Architecture of the nuclear pore complex coat, Stuwe [/bib_ref] [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] [bib_ref] Yeast Nup84-Nup133 complex structure details flexibility and reveals conservation of the membrane..., Nordeen [/bib_ref]. Bending at recognized 'hinge points' [bib_ref] Three-dimensional structure and flexibility of a membrane-coating module of the nuclear pore..., Kampmann [/bib_ref] [bib_ref] Atomic structure of the Y complex of the nuclear pore, Kelley [/bib_ref] [bib_ref] Architecture of the nuclear pore complex coat, Stuwe [/bib_ref] , we fit the Y complex (Extended Data [fig_ref] Figure 3 |: IR flexibility increases central channel size in native NPCs [/fig_ref]. It docks into both the CR and NR in a reticulated pattern of eight-membered rings (Extended Data . In each ring, we observed considerable density beyond the Y complexes. The additional density in the CR might correspond to Nup358, an expected component of this subassembly [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] (Extended Data . We also observed density protruding out of the CR towards the centre of the ring at the base of Nup85 (Extended Data . On the basis of previous cross-linking [bib_ref] Structure and function of the nuclear pore complex cytoplasmic mRNA export platform, Fernandez-Martinez [/bib_ref] [bib_ref] A short linear motif in scaffold Nup145C connects Y-complex with pre-assembled outer..., Teimer [/bib_ref] and cryo-EM/cryo-ET [bib_ref] Structure and gating of the nuclear pore complex, Eibauer [/bib_ref] [bib_ref] Structure of the cytoplasmic ring of the Xenopus laevis nuclear pore complex..., Huang [/bib_ref] studies, this density is probably occupied by the Nup214-Nup88-Nup62 complex. For the IR, we first docked the published composite model containing Nup93, Nup188/Nup205, Nup155 and the Nup62-Nup58-Nup54 complex 9 (Extended Data [fig_ref] Figure 3 |: IR flexibility increases central channel size in native NPCs [/fig_ref] , e). Although this model fits well, some elements appear to be shifted, suggesting that flexibility in the IR Nups may account for the larger diameter that we observed (Extended Data . To better fit the model, we isolated individual Nups and reoriented them locally to create an updated IR model (Extended Data . The NR is also composed of two eight-membered rings of Y complexes. As in the CR, we identified additional density near the Nup85 arms (Extended Data , and observed a linker density between the Y rings in agreement with a previous report 15 (Extended Data . Unique to the NR, we observed density at the base of the Y-complex-containing rings, which extends towards the IR (Extended Data , f). ## Asymmetric nuclear membranes at the npc Cross-sections through the cryo-ET map reveal that the NE at the NPC is asymmetric, with a steeper angle at the CR compared with at the NR (about 42° compared with about 28°, respectively), using the IR as a plane of reference . This membrane asymmetry is also evident in two-dimensional (2D) orthoslices of eight-fold symmetrized NPCs. In different planes of the NPC, it is readily apparent how the NE is shallower at the nucleoplasmic versus the cytoplasmic surface . ## Native npc has a larger central channel Compared with previous human NPC models, our map displays substantial differences, including larger IR and central channel diameters. Although the IR is essential for maintaining the architecture of the NPC central channel, it also anchors Nups with flexible, unstructured FG-rich domains that facilitate transport through the channel 7,28 . We found that the channel delineated by the IR has a volume that is about 75% larger than previously reported [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] [fig_ref] Figure 3 |: IR flexibility increases central channel size in native NPCs [/fig_ref]. We expect that these differences are due to the preserved cellular environment rather than any cell-type-specific differences, as we observed a similar architecture in HeLa-derived cells (Extended Data . Furthermore, we analysed data from mouse embryonic fibroblasts in which cryo-ETs of NPCs were captured by cryo-FIB milling or cell permeabilization followed by nuclease incubation [bib_ref] A lamin A/C variant causing striated muscle disease provides insights into filament..., Kronenberg-Tenga [/bib_ref]. The diameter of NPCs from cryo-FIB cells was again notably larger compared with the diameter of NPCs from permeabilized cells (Extended . In our IR model, Nup205/Nup188 (paralogues that are indistinguishable at this resolution), Nup155 and Nup93 are significantly shifted, whereas the Nup62-Nup58-Nup54 complex is stable (Extended Data . Propagated through the entire IR, these rearrangements explain how the central channel becomes larger. Adjacent IR subunits are nearly separated compared with previous models in which the subunits were snugly aligned [fig_ref] Figure 3 |: IR flexibility increases central channel size in native NPCs [/fig_ref] and Extended Data . The Nup93 and Nup205 subunits of adjacent IR structures form a pseudo-lock-and-key configuration in the constricted IR model; by contrast, here these components shifted to create gaps between the IR subunits [fig_ref] Figure 3 |: IR flexibility increases central channel size in native NPCs [/fig_ref]. In contrast to the IR, the diameters of the CR and NR are nearly identical to previous models (Extended Data . However, to accommodate a flatter overall assembly, the CR and NR rings flex in relation to the NE. At both the CR and NR, the Y-complex rings are angled more inward towards the central channel (Extended Data . ## Architecture of nup96-depleted npcs To better understand the contribution of each ring to the overall NPC assembly, we next analysed the structure of Nup96-depleted NPCs. We acquired 71 tomograms of cryo-FIB-milled NUP96::Neon-AID DLD-1 cells after auxin-induced Nup96 depletion and extracted 163 NPCcontaining subvolumes for subtomogram averaging (Extended Data . In two cells, we observed ONM herniations (Extended Data , indicating dysfunctional assembly of NPCs 30 . NPCs from this dataset could be divided into the following three classes : class 1, with all three rings (17% of particles); class 2, with NR-IR complexes (32%); and class 3, containing only the IR (51%). As we found no CR-IR complexes, this suggests that the CR is more readily removed from the NPC than the NR after Nup96 depletion. Furthermore, the diameters of each unique NPC assembly varied, and assemblies with NR-IR or IR only had significantly larger diameters (Extended Data . With the reduction from three to two to one ring, the spacing between the eight IR subcomplexes increases . In addition to the dilated central channel in IR-only NPCs, a cross-section analysis revealed changes in membrane orientations at the opening surrounding Nup96-depleted NPCs . The membrane has a smaller radius of curvature at the ONM-INM fusion, and the NE is more symmetric compared with in wild-type NPCs. # Discussion The architectural analysis of the NPC is a major challenge for structural biology. To date, the most accurate models of the human Article NPC are composite structures combining crystal or EM structures and structure-based models of subcomplexes with cryo-ET data of semi-purified, intact NPCs 9,15,31 . Our study reveals substantial NPC plasticity and shows that the purification of NEs influences the human NPC structure. We considered how the difference in IR diameter between in situ versus partially purified NPCs can be explained. As the NPC is integrated into the cellular matrix through the lamina and the cytoskeleton [bib_ref] Nonlinear mechanics of lamin filaments and the meshwork topology build an emergent..., Sapra [/bib_ref] [bib_ref] The molecular architecture of lamins in somatic cells, Turgay [/bib_ref] , it is conceivable that tensile forces acting on the NE could cause this change . Another consideration is that nuclear transport facilitated by FG-repeat extensions interacting with transport receptors may affect the NPC architecture, as reported in Xenopus oocytes [bib_ref] The nuclear import factor p10 regulates the functional size of the nuclear..., Feldherr [/bib_ref]. Furthermore, some FG-Nups stabilize the NPC scaffold [bib_ref] Natively unfolded FG repeats stabilize the structure of the nuclear pore complex, Onischenko [/bib_ref] ; there could therefore be a yet-to-be discovered connection. Modulating the IR diameter may be a general property of NPCs as we also observed this phenomenon in mouse embryonic fibroblasts, and it has been reported in Saccharomyces cerevisiae [bib_ref] In-cell architecture of the nuclear pore and snapshots of its turnover, Allegretti [/bib_ref] , Schizosaccharomyces pombeand human cells [bib_ref] Visualizing the molecular sociology at the HeLa cell nuclear periphery, Mahamid [/bib_ref] [bib_ref] Cone-shaped HIV-1 capsids are transported through intact nuclear pores, Zila [/bib_ref]. Moreover, we observed heterogeneity in the IR diameter among individual NPCs. Thus, structural variation is an intrinsic property of the NPC inside cells. IR flexibility could also be important for protein trafficking to the INM, which requires passage through the NPC while membrane anchorage is retained [bib_ref] In-cell architecture of the nuclear pore and snapshots of its turnover, Allegretti [/bib_ref]. A wider central channel not only has implications for the maximal size of soluble and membrane-bound cargo that can pass through the NPC, but it also changes the density of the FG repeats that fill the transport channel 39 . The influence of FG density on transport has been documented 40 . Our NPC model indicates that the FG density in the central channel is lower than previously thought, or that flexibility may be a means to modulate the FG barrier, consistent with suggestions for NPCs from Chlamydomonas reinhardtii 20 and S. pombe. In contrast to the IR diameter change, the CR and NR dimensions are similar to previous studies of semi-purified NPCs, suggesting that the three rings have some independence. Our findings in Nup96-depleted cells support a modular design, but also highlight the interdependence of these modules in regulating the central channel . This may indicate that the CR and NR act as molecular 'rulers' to define the upper limit of the IR dimensions, as proposed for C. reinhardtii 20 . However, the fact that the IR is malleable whereas the CR and NR are not also implies that the connections between the distinct rings (IR-NR and IR-CR, respectively) are flexible, explaining different assignments in various reconstructions [bib_ref] Molecular architecture of the inner ring scaffold of the human nuclear pore..., Kosinski [/bib_ref] [bib_ref] Integrated structural analysis of the human nuclear pore complex scaffold, Bui [/bib_ref] [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] [bib_ref] The human nuclear pore complex as revealed by cryo-electron tomography, Maimon [/bib_ref] [bib_ref] In-cell architecture of the nuclear pore and snapshots of its turnover, Allegretti [/bib_ref] [bib_ref] Structure and gating of the nuclear pore complex, Eibauer [/bib_ref]. Our model also exposes the asymmetry of the CR and NR substructures and their effect on the NE, as previously observed in the Xenopus laevis NPC 26 . Furthermore, we observed asymmetry in the nuclear membrane at the NPC, suggesting that the membrane connections between the NR and CR are different. It is possible that ring-specific Nups make interactions with the cytoskeletal/laminar network or with ONM/INM-specific proteins to bring about this asymmetry. Alternatively, the ring-linking densities (such as Nup358 at the CR or the unassigned NR linker) could contort the CR/NR reticulated ring structures differentially, as their diameters are slightly different. Thus, the tension that each ring might impose on the NE may also lead to this asymmetry. Flexibility of the NPC emerges as a major challenge for achieving true high resolution. In addition to flexibility, architectural heterogeneity might come from NPCs that are assembled from different subsets or stoichiometries of Nups 41,42 . Thus, the structure described here probably represents just one of several NPC states that are still to be discovered. ## Online content Any methods, additional references, Nature Research reporting summaries, source data, extended data, supplementary information, acknowledgements, peer review information; details of author contributions and competing interests; and statements of data and code availability are available at https://doi.org/10.1038/s41586-021-03985-3. Publisher's note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. ## © the author(s) 2021 ## Article # Methods Cell culture and Nup96 depletion NUP96::Neon-AID DLD-1 cellswere grown in standard tissue culture conditions (37 °C with 5% CO 2 ) using DMEM medium supplemented with 4.5 g l −1 glucose and 4.5 g l −1 sodium pyruvate (Corning), 10% FBS (Gemini Bio) and 2 mM GlutaMAX (Gibco). Next, cells were cultured for 18-24 h on freshly glow-discharged carbon-coated EM gold grids (200 mesh, R1.2/1.3 or R2/1; Quantifoil Micro Tools) before plunge-freezing. For Nup96 depletion, a 250 mM auxin (3-indoleacetic acid, Sigma-Aldrich) solution was added to the cells to a final concentration of 1 mM, followed by incubation at 37 °C for 4-8 h before plunge-freezing. HeLa-derived (TOR1A/TOR1B/TOR3A-knockout) cells [bib_ref] The AAA + ATPase TorsinA polymerizes into hollow helical tubes with 8.5..., Demircioglu [/bib_ref] were cultured in the same conditions but never subjected to auxin treatment. ## Cryo-fib milling of lamellae from dld-1 cells To prepare lamellae, we used both an Aquilos FIB-SEM system (Thermo Fisher Scientific) and a Crossbeam 540 (Zeiss) with cryo stage (Leica) using similar methods as previously described [bib_ref] Preparing samples from whole cells using focused-ion-beam milling for cryo-electron tomography, Wagner [/bib_ref]. On the Aquilos system, grids were loaded into the sample chamber and sputtered with an initial platinum coat (15 s) followed by a second organometallic platinum protective layer using the gas injection system (GIS, 15 s). Samples were tilted to an angle of 15°, and FIB ablation of cellular material was performed in a stepwise manner by focusing the gallium beam at 30 kV on parallel rectangular patterns with overand under-tilting to the desired thickness as follows: (1) (7) 15°, 30 pA, gap = 150 nm. The initial width of the lamellae was set to 13 µm but, at each reduced current, we reduced the width by 0.25 µm, for lamellae with a final width of 11-12 µm. Expansion/relief joints to reduce tension at the lamella were milled 3 µm away from the sides of the lamella at step 1 with a 1-µm-wide pattern. A similar procedure was used on the Crossbeam 540 FIB-SEM. Grids were first electron-beam-coated with 2 nm of platinum in an ACE 900 system (Leica) before being loaded into the Crossbeam sample chamber with a VCT 500 (Leica) vacuum transfer system for organometallic platinum coating. FIB ablation was performed using only four stepwise currents and without over-and under-tilting as follows: (1) 700 pA, gap = 3 µm; (2) 300 pA, gap = 1 µm; (3) 100 pA, gap = 400 nm; and (4) 50 pA, gap = 150 nm. In total, we prepared 37 lamellae from non-auxin-treated NUP96::Neon-AID DLD-1 cells, and 115 lamellae from auxin-treated cells to obtain our data. ## Tilt series acquisition and processing Cryo-ET datasets were acquired on a Titan Krios G3i operating at 300 keV equipped with a BioQuantum post-column energy filter and a Gatan K3 direct electron detector. Tilt series were acquired at a magnification of ×26,000, resulting in a pixel size of 3.4 Å at the specimen level using Tomo v.5.3.0 (Thermo Fisher Scientific). We used a tilt range of −52° to +68°, starting at +8° with a bidirectional scheme 45 resulting in a total dose of about 145 electrons per angstrom squared. Tilt series of HeLa cells (TOR1A/TOR1B/TOR3A-knockout) [bib_ref] The AAA + ATPase TorsinA polymerizes into hollow helical tubes with 8.5..., Demircioglu [/bib_ref] were acquired with an accumulative dose of about 120 electrons per angstrom squared. Defocus values varied between −5.0 µm to −2.5 µm through the entire data acquisition process. Representative tomograms are included . Tilt series were aligned with 4× binned projections using patch tracking in the IMOD software package [bib_ref] Computer visualization of three-dimensional image data using IMOD, Kremer [/bib_ref]. When available, contaminations generated by the FIB process were used as fiducial markers. The contrast transfer function (CTF) was determined and corrected as described previously [bib_ref] Structure and gating of the nuclear pore complex, Eibauer [/bib_ref] , and is summarized here. The mean defocus was estimated by strip-based periodogram averaging for each tilt series. Using the mean defocus, the tilt angle and axis orientation, the defocus gradient for each projection was determined. CTF correction using the defocus gradient was then performed by phase-flipping each projection image. CTF-corrected stacks were next dose-filtered using the IMOD mtffilter function [bib_ref] Computer visualization of three-dimensional image data using IMOD, Kremer [/bib_ref]. NPC coordinates were picked manually using IMOD. During NPC particle picking, the relative orientation of the NPC to the NE was determined to prealign the NPCs. Next, subtomograms of NPCs were reconstructed using IMOD. Detailed imaging parameters are summarized in Extended Data . ## Subtomogram averaging For the wild-type DLD-1 structure, 194 prealigned NPCs were further aligned using iterative missing-wedge-weighted subtomogram alignment and averaging with the TOM toolbox (tom_corr3d) [bib_ref] Retrovirus envelope protein complex structure in situ studied by cryo-electron tomography, Forster [/bib_ref]. Half-set averages were merged after each iteration and used as the template for the next iteration. First, full NPCs were aligned using 8× binned subvolumes and applying eightfold-fold symmetry. This step was repeated using 4× binned subvolumes. Subsequently, these alignments were used to extract the eight asymmetric units (1,552 protomers) for each NPC according to the eight-fold symmetry as previously described [bib_ref] Snapshots of nuclear pore complexes in action captured by cryo-electron tomography, Beck [/bib_ref]. Twice-binned full protomers were aligned and further refined by applying masks for the individual rings (CR, IR and NR). Next, subprotomers were extracted based on the previous alignment step. At this stage, single protomers were manually inspected and missing protomers from incomplete NPCs, misaligned protomers and protomers with a low signal-to-noise ratio were excluded. After this step, 1,252 protomers remained for the subsequent steps. Newly extracted subprotomers were further aligned. Resolution was measured using the 0.5 criterion and soft masks to exclude an artificial contribution to the measured resolution using the Electron Microscopy Data Bank (EMDB) validation server (https://www.ebi.ac.uk/pdbe/emdb/validation/fsc/). Subprotomer volumes were B-factor sharpened using Relion 49 with a B-factor of −2,000 Å 2 . The final full NPC model was generated by fitting the subprotomer volumes into the full NPC average using UCSF Chimera [bib_ref] UCSF Chimera-a visualization system for exploratory research and analysis, Pettersen [/bib_ref]. Averaging of auxin-treated (Nup96-depleted) data was performed similarly. First, 163 full NPCs were aligned using 8× binned subprotomers and applying eight-fold symmetry. Next, NPCs were classified by manually inspecting the subvolumes into three classes containing CR, IR and NR , IR and NR (53 NPCs), or IR only (83 NPCs). These three classes were further aligned using 8× binned subvolumes at full NPC level using eight-fold symmetry. For the HeLa (TOR1A/TOR1B/TOR3A-knockout) average, 27 NPCs were aligned similar to the wild-type dataset but without dose-filtering of the stacks. After protomer extraction, 36 protomers were excluded and protomers were aligned. The final map (180 protomers) was generated by fitting the protomer average back into the full NPC map using Chimera. ## Model fitting To create models for the Y complexes in the CR and NR, we performed unbiased global fitting using structural models derived from previously reported human NPC structures [bib_ref] Molecular architecture of the inner ring scaffold of the human nuclear pore..., Kosinski [/bib_ref] [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref]. For these complexes, we began with the previously reported model and performed a global fitting analysis as implemented in UCSF Chimera 50 . The fitting was performed independently for the CR and NR using a three-protomer segmented model as our single protomers do not necessarily contain a complete protomer of the eight-fold symmetric NPC (that is, a single protomer may contain half of the Y complex from adjacent protomers). For the IR, a single protomer was used as it contained the entire complex. All fitting runs were performed using Chimera and 1,000,000 random initial placements and local cross-correlation (Chimera's correlation about the mean (CAM)), or a combination of local cross-correlation and overlap (CAM + OVR) was used when local cross-correlation did not provide statistically significant fits (as others have observed). We computed fit scores using both metrics for each model but, for the IR, the local cross-correlation (CAM) provided statistical significance to our model, whereas, for the Y complex, the combination metric (CAM + OVR) provided statistical significance. We noted the scores and adjusted P values (described below) on the figure for each scoring metric. For each fitting run, the statistical significance was assessed as a P value that was calculated from normalized fitting scores. To calculate the P values, we transformed the CAM or combined CAM + OVR scores into Z scores, derived a two-sided P value for each fit and then corrected the P values for multiple testing using the conservative Benjamini-Hochberg procedure. This workflow was performed using a Python script running SciPy.Stats (for P value and Z-score analysis) [bib_ref] SciPy 1.0: fundamental algorithms for scientific computing in Python, Virtanen [/bib_ref] , the StatsModels module (for Benjamini-Hochberg analysis) [bib_ref] statsmodels: econometric and statistical modeling with Python, Seabold [/bib_ref] and Matplotlib (for plots) [bib_ref] Matplotlib: a 2D graphics environment, Hunter [/bib_ref]. For the IR complex, we identified a significant fit in the expected position as described in the most recent human structure (Protein Data Bank (PDB): 5IJN) [bib_ref] Molecular architecture of the inner ring scaffold of the human nuclear pore..., Kosinski [/bib_ref] , although it was obvious that some of the Nups in this model could be shifted to better fit our density map. We therefore followed a previously described approachand optimized the fits by local refitting of individual subunits or domains. For this refinement, we used the option in Chimera to use a map simulated from atoms at a resolution of 25 Å and optimized the fitting for correlation. Using this approach, we fit subcomplexes made of the chains F, G, H, X, Y and Z (two copies of Nup54, Nup58 and Nup62), L, M, N, R, S and T (two additional copies of Nup54, Nup58 and Nup62), each copy of Nup205 (chains D, J, P and V), and the four copies of Nup93 (chains C, I, O and U). To fit the Nup155 chains, we subtracted densities with a radius of 10 Å of the fitted models above from the determined NPC map. In this difference map, we fit the individual Nup155 copies into the map using the procedure described above (chains E, Q, K, W). The Nup155 chains A and B of 5IJN could not be fit into our map reliably. We next ran a new unbiased global search using this model as the input for completeness. For the Y complex, we identified a significant fit in the same position as described in the most recent human NPC structure CR and NR (PDB: 5A9Q) [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] using a double Y complex as the reference molecule. As the 5A9Q model contains gaps in the heterotetrameric core element of the Y complex called the 'hub' (Nup160, Nup85, Nup96 and Sec13), we decided to replace the hub of the 5A9Q model with the hub of a new composite model that we created using published crystal structures and threading models. We first generated an S. cerevisiae composite model combining a previous structure of the Y complex hub (Nup120-Nup145N-Sec13-Nup85) [bib_ref] Architecture of the nuclear pore complex coat, Stuwe [/bib_ref] (PDB: 4XMM) and the Nup84-Nup133 subcomplex 24 (PDB: 6X02). This S. cerevisiae Y-complex composite was then used to template the human homologue, using published structures and threading models. The hub of this updated model was then used to rigid-body dock into the CR/NR of our map at the same position that the 5A9Q hub occupied, and the Nup107-Nup133 subcomplex from 5A9Q was kept as is, creating a new complete Y-complex assembly. Fitting was further refined by cutting Nup160 between residue 933 and 934 (ref. [bib_ref] Atomic structure of the Y complex of the nuclear pore, Kelley [/bib_ref] and fitting the N-terminal part of Nup160 (Nup160-N) together with Nup37 as a rigid body into our map. The C-terminal part of Nup160 was fit as part of the hub. The CR model was then also placed for the inner Y ring and not further modified. Fitting for the NR Y complex was performed similarly. For the outer Y ring, only the hub and the Nup107-Nup133 subcomplex were separated and fit as individual rigid bodies. For the IR of the NR Y complex, the model without Nup160-N and Nup37 was fit into the map. For fitting of the Nup160-N-Nup37 subcomplex, we subtracted the densities of previously fit models with a radius of 20 Å from the map and fit the subcomplex into the remaining density. After creating the final models for Y complexes in both the CR and NR, we performed another round of unbiased global fitting and identified a similar location at which the Y complex of 5A9Q docked. A final local optimization run maximized density overlap. We strongly urge that the final fits are not interpreted at atomic resolution. Instead, our fitting simply aids in assignment of our density toward understanding how each of the subcomplexes is positioned. The IR model described above was placed manually into the centre of the IR protomers of the subtomogram maps generated from auxin-treated (Nup96-depleted) cells. ## Visualization Visualizations were performed using UCSF Chimera 50 . Representative tomograms of DLD-1 cells were reconstructed in 8× binned, 12× SIRT-like filtered tomograms in IMOD [bib_ref] Computer visualization of three-dimensional image data using IMOD, Kremer [/bib_ref]. Snapshots of mouse embryonic fibroblast (MEF) NPCs were recorded after manually aligning the NPCs in IMOD Slicer. The orthoslice views of the full-NPC average, wild-type map and auxin maps were taken using tom_volxyz. Scripts were implemented in MATLAB (MathWorks) and using the tom_toolbox [bib_ref] TOM software toolbox: acquisition and analysis for electron tomography, Nickell [/bib_ref]. Local resolution analysis was performed as previously reported [bib_ref] Structure and gating of the nuclear pore complex, Eibauer [/bib_ref] , but also summarized here. To calculate the local resolution of the subprotomers, the full subprotomer volume (100 × 100 × 100 voxels) was divided into smaller subvolumes (box size, 40 × 40 × 40) along a regular spacing of 4 × 4 × 4. Resolution was measured between the subvolumes of the two half sets and using the 0.5 threshold criterion. Subvolumes were masked with a spherical mask prior to FSC calculation. Data points between measured values were interpolated and visualized using Chimera's Surface color function. # Npc diameter analysis For diameter measurements, single NPCs were measured using orthoslices at the level of the pore membrane. For the DLD1 and HeLa datasets, aligned NPCs that would proceed to full-NPC averaging in this study were used. For the MEF datasets [bib_ref] A lamin A/C variant causing striated muscle disease provides insights into filament..., Kronenberg-Tenga [/bib_ref] , NPCs were manually aligned in IMOD slicer and measured. Distance was measured between the NE at the narrowest point of each NPC manually. When possible, measurement was performed in two orthogonal directions and the average was calculated. Otherwise, only a single measurement was performed per NPC. When measurement was not possible because of strong misalignment or a poor signal-to-noise ratio, no diameter was measured. Scatter plots for NPC diameter analysis were created using Prism 9 (GraphPad). ## Statistics and reproducibility Representative micrographs are provided in and Extended Data , f. The micrograph in highlights the three-ring NPC architecture directly visualized in cryo-ET and was chosen from the dataset of 54 wild-type DLD-1 cell tomograms, all of which reproducibly show a three-ring architecture. The micrograph in Extended Data was chosen from the auxin-depleted DLD-1 cell dataset (71 tomograms), and this image was specifically chosen because it highlights single-ring NPCs that we describe in this Article. Finally, the micrograph in Extended Data was specifically chosen to provide the reader with an anecdotal observation that we identified only twice in the dataset (2 out of 73 tomograms) and these tomograms were excluded from subsequent processing (71 tomograms were used for subtomogram averaging). ## Reporting summary Further information on research design is available in the Nature Research Reporting Summary linked to this paper. ## Data availability Cryo-EM maps for the human DLD-1 NPC have been deposited in the EMDB with the following accession codes: EMD-12811 (CR), EMD-12812 (IR), EMD-12813 (NR) and EMD-12814 (full composite NPC). Coordinate files for the CR, IR and NR docked complexes have been deposited in the PDB with the following accession codes: 7PEQ (CR and NR complex) and 7PER (IR complex). Representative tilt series of lamella from DLD-1 cells have been deposited in the EMDB under accession code EMPIAR-10700 (wild-type, non-depleted cells) and EMPIAR-10701 (Nup96-depleted cells). Density maps for the human NPC from isolated nuclei used for comparison in this study can be found at the EMDB with accession number EMD-3103 (ref. [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] , as well as the recent NPC from HIV-infected T cells with accession number EMD-11967 (ref. [bib_ref] Cone-shaped HIV-1 capsids are transported through intact nuclear pores, Zila [/bib_ref]. Moreover, models Extended Data | Details of in situ human NPC architecture. a, Orthoslices through the nucleocytoplasmic axis, CR, and NR for our cryo-ET map of the human NPC from FIB-milled DLD-1 cells. Slice thickness: 3.4 nm. b, Fourier shell correlation curves of the CR, IR, and NR regions in our map. FSC 0.5 and FSC 0.143 are indicated as dotted lines. c, The local resolution for the CR, IR, and NR protomers is visualized as surface color. d, Segment of the CR to highlight fitting of the Y complex structure and several unique features including putative Nup358 density (green arrowheads), extra unassigned density attached to the inner ring of the Y complex Nup85 (black arrowheads), and density we attribute to the Nup214 complex (blue arrowheads). e, Segment of the IR from our cryo-ET density with updated model fit inside. f, Segment of the NR to highlight fitting of the Y complex structure and several unique features including linker density between the Y complex rings (red arrowheads), unassigned density attached to the inner ring of the Y complex Nup85 (black arrowheads), and linkers between the Y complex inner ring and the IR (purple arrowheads). | Comparison of NPC structures from purified NEs and cryo-FIB suggests the cellular environment impacts NPC architecture and IR diameter. a, Comparison of human NPC density maps from cryo-FIB and nuclear envelope purification techniques. Measurements for the membrane-to-membrane and IR central channel diameters, as well as the height across the NE are shown. Interestingly, an asymmetric membrane curvature may also exist at human NPCs in HIV-infected cells [bib_ref] Cone-shaped HIV-1 capsids are transported through intact nuclear pores, Zila [/bib_ref] , though the angles of the membranes appear different than ours. We refrain from making any speculations about this asymmetry because the deposited map comes from cells infected with HIV-1 virus. b, Measurements of NPC diameter from single NPCs in DLD-1 cells (non-auxin treated) as well as HeLa cells (TorsinA/B/3A KO), both prepared by cryo-FIB. Median values from each distribution are indicated as a dotted line and below the plot. **** indicates a two-tailed p-value ≤ 0.0001 from Mann-Whitney U analysis. c, Measurements of NPC diameter from single NPCs in MEFs using cryo-FIB or nuclear envelope purification techniques. Median values from each distribution are indicated as a dotted line and below the plot. Representative tomographic slices from intact cells using cryo-FIB milling and from isolated NE preparations are shown. Slice thickness: 8.9 nm. [fig_ref] Figure 3 |: IR flexibility increases central channel size in native NPCs [/fig_ref] | See next page for caption. | Updated model for native IR. a, Side-by-side comparison of the previous IR model (PDB: 5IJN) with our updated model to fit the DLD-1 NPC map. b, Superimposition of our updated IR model with the previous model shows the rearrangements required to fit our density map. Each pane highlights a different nucleoporin colored in our updated model as in (a), or colored dark grey from the previous model. c, Views of three updated IR-models in our map compared with the previous model and human NPC map. d, Same as (c) but with only models. e, Cross-section views of the NR in our map at both high and low threshold to show connectivity with the IR. f, Same as (e), though no density can be observed between the CR-IR, likely due to flexibility. Flexibility could explain why the ring-connecting densities, proposed to be Nup155 9 , remain conspicuously poorly resolved in our map compared to the previous human maps [bib_ref] Integrated structural analysis of the human nuclear pore complex scaffold, Bui [/bib_ref] [bib_ref] In situ structural analysis of the human nuclear pore complex, Von Appen [/bib_ref] [bib_ref] The human nuclear pore complex as revealed by cryo-electron tomography, Maimon [/bib_ref]. We hypothesize that this observation may be unique to the more "constricted" IR state observed previously, locking the ring-connecting proteins into a more rigid state. In the same vein, others have shown that NPCs from yeast, both semi-purified 36 and in their native state [bib_ref] In-cell architecture of the nuclear pore and snapshots of its turnover, Allegretti [/bib_ref] , can contain multiple flexible linkers between the outer rings and IR, suggesting this arrangement could provide resilience to the NPC structure. | Details of Nup96-depleted NPC structures. a, Tomographic slice from a lamella prepared by cryo-FIB of NUP96::Neon-AID DLD-1 cells in the presence of auxin for 4 h (Nup96-depleted). Arrowheads indicate Nup96-depleted NPCs. b, Orthoslices across the nucleocytoplasmic axis for the final aligned maps of the three Nup96-depleted NPC classes. c, Measurements of NPC diameter from single NPCs in each of the data sets as well as the non-auxin treated (WT). *** indicates a two-tailed p-value ≤ 0.001 and **** indicates a two-tailed p-value ≤ 0.0001 from Mann-Whitney U analysis of each distribution compared with the wild-type condition. Median values from each distribution are indicated as a dotted line and shown above the plot. d, Orthoslices across the nucleocytoplasmic axis of single NPCs in each of the three Nup96-depleted NPC classes. Slice thickness is 4.1 nm. e, Orthoslices across the nucleocytoplasmic axis of class averages after manual orientation alignment and classification. f, Fourier shell correlation curves for the Nup96-depleted NPC classes. FSC 0.5 and FSC 0.143 are indicated as dotted lines. g, Tomographic slice of Nup96-depleted cells showed instances of NE herniation at the ONM (example indicated by arrow). Slice thickness for all panels except d: 2.7 nm. ## Extended data ## Extended data ## Extended data ## Extended data Corresponding author(s): Medalia, O. and Schwartz, T.U. Last updated by author(s): Reporting Summary Nature Portfolio wishes to improve the reproducibility of the work that we publish. This form provides structure for consistency and transparency in reporting. 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Representative tilt series of lamella from DLD-1 cells have been deposited in the EMDB under accession code EMPIAR-10700 (wild-type, non-depleted cells) and EMPIAR-10701 (Nup96-depleted cells). [fig] Figure 1 |Figure 2 |: The NPC structure from human DLD-1 cells. a, Tomographic slice from a lamella of a NUP96::Neon-AID DLD-1 cell in the absence of auxin (wild-type condition). The slice thickness is 2.7 nm. The arrowheads indicate each of the NPC rings. The arrow indicates an adjacent NPC. Scale bar, 100 nm. b, Whole NPC map from DLD-1 cells. Scale bar, 20 nm. c, Cross-section of a cryo-ET map with fitted structures for the CR (blue and yellow), IR (orange and magenta) and NR (light blue and gold). The central channel diameter (57 nm), the membrane-to-membrane diameter (105 nm) and the height across the NE (37 nm from IR-CR, 29 nm from IR-NR) are indicated. Asymmetric nuclear membrane at the NPC. a, Cross-section of the NPC structure. The angles indicate measurements from the membrane distal edge of the IR to the edge of the nuclear membrane at the NR or CR. b, 2D orthoslices through the full-NPC average. Top, NPC map; the planes of the 2D slices (bottom) are indicated. Scale bar, 20 nm. Plane 1, at the point of the interaction between the CR Y complex and the membrane. Plane 2, at the point of the interaction between the NR Y complex and the membrane. Plane 3: sSlice in which no Y complexes touch membrane. The slice thickness is 1.4 nm. [/fig] [fig] Figure 3 |: IR flexibility increases central channel size in native NPCs. a, Segmentation of the IR from our model and the previous human NPC model. The IR diameter (membrane to membrane) and central channel diameter measurements are indicated. The IR channel volume was calculated as a cylinder with a height of 20 nm. b, Top view of three IR subunits in our model compared with the previous model. c, Schematic showing the lock-and-key-like architecture of the previous model and how the IR subunits have shifted in our model. [/fig]

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Concepts of functioning and health important to people with systemic sclerosis: a qualitative study in four European countries Objective To describe the experiences of people with systemic sclerosis (SSc) in different European countries of functioning and health and to link these experiences to the WHO International Classifi cation of Functioning, Disability and Health (ICF) to develop a common understanding from a bio-psycho-social perspective. Method A qualitative multicentre study with focusgroup interviews was performed in four European countries: Austria, Romania, Sweden and Switzerland. The qualitative data analysis followed a modifi ed form of 'meaning condensation' and the concepts that emerged in the analysis were linked to the ICF. Results 63 people with SSc participated in 13 focus groups. In total, 86 concepts were identifi ed. 32 (37%) of these were linked to the ICF component body functions and structures, 21 (24%) to activities and participation, 26 (30%) to environmental factors, 6 (7%) to personal factors and 1 (1%) to the health condition itself. 19 concepts (22%) were identifi ed in all four countries and included impaired hand function, household activities, paid work, drugs, climate and coldness, support from others and experiences with healthcare institutions, nonpharmacological treatment, social security and benefi ts. Conclusion Concepts identifi ed in all four countries could be used for guiding clinical assessment, as well as interdisciplinary team care and rheumatological rehabilitation for patients with SSc. For a full understanding of the aspects of the disease that were most relevant to people with SSc, people with SSc from multiple countries needed to be involved. # Introduction Systemic sclerosis (scleroderma; SSc) is a systemic, infl ammatory and autoimmune disorder characterised by the excessive deposition of extracellular matrix in the skin and in inner organs which can lead to a wide range of organ damage. [bib_ref] Immunopathogenesis of systemic sclerosis, White [/bib_ref] Based on the amount of skin affected, a limited and a diffuse form of SSc are distinguished. Skin thickening occurs earlier in diffuse disease and severe skin involvement is a predictor of later organ involvement and outcome. Skin induration as well as joint and muscle involvement often lead to a progressive reduction in the range of motion. Up to 90% of patients with SSc report loss of hand grasp ability related to factors such as skin sclerosis of fi ngers, ischaemia with ulcers, arthralgia, calcifi cations and wrist extension reduction leading to severe hand disability. These may be signifi cant factors for diffi culties in performing activities of daily living. SSc affects both physical and mental health-related quality of life, [bib_ref] Canadian Scleroderma Research Group . Health-related quality of life in systemic sclerosis:..., Hudson [/bib_ref] which may cause problems of functioning and restriction in participation in society. It may thus be essential for optimal patient care to understand functioning from a bio-psycho-social perspective, which is currently best represented in the framework and model of the WHO International Classifi cation of Functioning, Disability and Health (ICF).The ICF comprises several categories ranging from body functions and structures, activities and participation to contextual factors. According to these categories, problems of patients with a certain disease can be classifi ed. If a patient reports problems with fi ne motor hand function, this may be linked to the corresponding ICF category (d440fi ne hand use) and can thus be 'translated into ICF language'. Based on this 'translation', problems of patients with a certain disease can be described in a frame of reference and compared with those of other patients, of other countries, of other diseases and so on. ICF categories are worded in neutral language, so that a certain category can either be a 'problem' or a 'resource'.In SSc, no data were available on functioning in daily life of people from different European countries from such a comprehensive, bio-psycho-social perspective. Qualitative methodology provides the possibility of exploring the perspective of those who experience a disease. In SSc, only a few previous studies have used a qualitative research approach. The aim of this study was to describe the experiences of people with SSc in different European countries of functioning and health and to link these experiences to the WHO ICF to develop a common understanding from a bio-psycho-social perspective. # Methods design A qualitative focus-group interview study was performed in the following four European countries: Austria (A), Romania (R), Sweden (S) and Switzerland (CH). ## Participants People with SSc were recruited from the outpatient departments of the participating centres. Patients who were eligible had to meet the American College of Rheumatology criteria for SSc,were asked whether they would like to participate in the study and were fully informed about the study procedures. Patients who were willing to participate gave written informed consent according to the Declaration of Helsinki. The study was approved by the institutional review boards and ethics committees of the participating centres. Sampling of patients in each country followed a maximum variation strategy 20 based on the following criteria: age, sex, disease duration and diffuse/limited SSc. ## Sample size Two to four focus groups were conducted in each country. From earlier studies, we estimated that about 8-10 focus groups were needed to obtain data of suffi cient depth and breadth. Qualitative research typically uses small sample sizes with a diverse range of participants. In this perspective, individual experiences which can express variations of a given phenomenon and potentially raise new and unexpected issues are more important than numbers and frequencies. 23 ## Data collection All focus groups were chaired by a trained and experienced moderator together with one assistant responsible for observing the group and recording the data. The fi ve questions for the focus groups were based on the following ICF domains: body functions (eg, "If you think about your body and mind, what does not work the way it is supposed to?") and structures, activities and participation, environmental and personal factors. Negative and positive aspects were dealt with by, for example, referring to barriers, facilitators and resources. According to a predefi ned protocol in an earlier study, 23 the questions were translated, back translated and the translations analysed so that a fi nal version could be established for the participating countries. Each group involved up to seven people. A short introduction including the purpose of the study was given in lay terms to all patients at the beginning of each focus group. The focus-group interviews were tape-recorded and transcribed verbatim. # Data analysis Qualitative data analysis followed a modifi ed form of 'meaning condensation',which involved the following steps: fi rst, the transcribed interviews were read through by the principal investigators in each country (AB, CM, MM/CB, JS) to obtain an overview of the collected data. Second, the data were divided into 'meaning units', which were defi ned as specifi c units of text, either a few words or a few sentences with a common meaning.Third, the concepts contained in the meaning units were identifi ed. A meaning unit could contain more than one concept. Not all concepts had to be on the same level (could be either lower-level or higher-level concepts) with the same range of meanings and attributes. So lower-level concepts could be grouped together under a higher-level concept, which had then to cover the whole content and attributes of the lower-level concepts. An example is described in fi gure 1 . When these analyses had been made in each country, the concepts were translated into English. Then, the concepts from all countries were combined (TS) using the framework of the ICF-for example, concepts from all countries relating to the ICF domain body functions were grouped under this domain and compared with each other for similarities and differences. Lower-level concepts (no matter which country) were grouped under higher-level concepts (from the same or any other country). An example was the higher-level concept 'emotional issues', which fi nally included the following lower-level concepts: anxiety about next possible symptoms and fl ares, depression, feeling sad, resignation, hope (from Austria); anxiety, emotional vulnerability, depressive mood, unhappiness (from Romania); emotional infl uence, downcast, depressed (from Sweden); feeling of depression, anxiety about losing a job, anxiety about losing one's independence (from Switzerland). In this process, the researchers (the fi rst author together with at least one researcher from each country) went back and forth between possible higher-and lower-level concepts and checked the scheme of concepts for each country so that meanings assigned would not be lost. In the fi nal step of the analysis, all concepts grouped under a specifi c ICF domain were linked to the corresponding ICF categories within this domain according to predefi ned rules [bib_ref] ICF linking rules: an update based on lessons learned, Cieza [/bib_ref] : the concept 'emotional issues', for example, was linked to a corresponding ICF category called 'b152-emotional functions'. Concepts were linked to the most specifi c ICF category. If concepts were more general and no specifi c ICF category was available, then they were linked to ICF chapters-for example, 'chapter d5-self-care' in the component 'activities and participation'. Concepts related to personal factors were linked to ICF whole component 'personal factors' (PF) because this ICF component does not include separate categories. Concepts related to the disease itself were linked to 'health condition' (HC). In addition, we counted the concepts for each country and for each ICF component partly following the approach described as 'meaning categorisation' by Accuracy and rigor of the qualitative analysis All local principal investigators (AB, CM, MM/CB, JS) had extensive experience with qualitative research before this project. Two pages of a transcript translated into English were sent round and each investigator extracted meaning units and the concepts contained in these meaning units before starting to analyse the local data. The results of this two-page exercise were discussed together to obtain a common understanding about the meaning units and depth of concepts until consensus was Linking to the ICF: b152 emotional functions -'b' is referring to body functions and structures An example of the qualitative analysis: a meaning unit, a lower-level concept extracted from this meaning unit and the corresponding higher-level concept and ICT category. achieved. Before a fi nal scheme of concepts was established, the concepts grouped under a certain ICF domain were sent back to the local investigators for evaluation and feedback. According to the suggestions of the local investigators, the concepts were then refi ned. # Results participants In total, 63 people with SSc participated in 13 focus groups (four in Austria, two in Romania, four in Sweden and three in Switzerland). Characteristics of the participants of each country are presented in table 1 . The number of participants in each focus group ranged from three to eight. ## Concepts obtained in the qualitative analyses The local investigators extracted 181 concepts in Austria, 99 in Romania, 159 in Sweden and 114 in Switzerland. Most concepts from a certain country showed similarities to the concepts derived from the qualitative data in the other countries-for example, the concepts 'problems with fi ngers in daily activities' from Austria were considered to be similar to 'functions of hands and arms and fi ne motor functions' from Romania, 'impaired hand function' from Sweden and 'impaired fi ne motor function of hands' from Switzerland. In all countries, the participants stated that SSc infl uenced and affected functioning and health. The ICF component to which the largest number of concepts was linked was body functions and structures (32 (37%) of the total number of 86 higher-level concepts). These concepts were also related to activities and participation and environmental and personal factors. A participant described this in the following way at the start of the group session: In my point of view everything is spinning around the same phenomenon, which for me was and still is . . . a total upsidedown turn of everyday life. And this is mostly because of the psychological manifestations (body function), not only because of physical problems (body function). What bothers me most? Of course the symptoms of the Raynaud's syndrome bother me, the fact that my hands become blue (body function), the fact that I don't look at them when I go out (activities and participation) in cold weather (environmental factor), the fact that I look at the hands of all people. But mostly the psychological issues (body functions) bother me. Some participants considered physical symptoms to be related to psychological functions (body functions), to activities and participation (eg, to go out), as well as to environmental factors (eg, climate). A participant expressed this relationship in the following way: If it's 20 degrees below zero outside, you don't go out at all . . . then I was really depressed, because it was so cold for such a long time. I didn't go out to get my mail from the mail box for almost three weeks. [fig_ref] Table 2: Concepts mainly related to the ICF components Applying self-treatment strategies, such as... [/fig_ref] shows the scheme of higher-level concepts derived from the qualitative data analyses of all countries. Nineteen concepts (22% of all 86 concepts) were identifi ed in all four countries (marked in grey in the table 2 ). Of these, nine were body functions and structures, three activities and participation and two personal factors. Within the environmental factors, fi ve concepts ('drugs and side effects of these', 'climate, cold weather, too much heat, cold in one's fl at', 'support of partner, family, relatives, friends', 'experiences with healthcare institutions including negative experiences' and 'non-pharmacological treatment. . . healthcare institutions, social security system, benefi ts' ( table 2 )) were found in all four countries, whereas most other concepts were identifi ed in one or two countries only. In the area of body functions and structures, activities and participation and personal factors, most concepts were found in at least two or three countries. ## Comparison of concepts from all countries In total, 23 concepts (27% of all 86 concepts) emerged in one country only. Of these, 10 were considered to be body functions and structures by the researchers, three activities and participation and 10 environmental factors. An example is the concept 'affected sex life' which was mentioned in one country, but not in any of the other countries. A participant described 'affected sex life' in the following way: I can talk about it-perhaps it is a stupid subject to raise-perhaps it is not, but my sex life with the cytotoxic drugs . . . I don't know if everyone reacts to cytostatics in such a way . . . but I have lost the urge for things like that . . . Later in the same focus group another participant expressed: My partner dislikes that I'm tired and don't want to have sex, he thinks there's something wrong with him. Some participants expressed the feeling that environmental factors could be both facilitators and barriers. While experiences with healthcare institutions were found to be supportive by some people with SSc ( table 2 ), others had had negative experiences with healthcare institutions: 'took a long time until diagnosis', 'wrong treatment of uninformed professionals', 'diffi culty to get an appointment' and 'no one knows what to do against dryness of mucosa'. Also in the area of body functions and personal factors, positive and negative aspects could be part of one concept. An example was the concept 'emotional issues' which included 'depression', 'anxiety', 'resignation', but also 'hope'. # Discussion This study described concepts important to people with SSc obtained in a qualitative focus-group study in four European countries. As far as we know, this is the fi rst multicentre study on the experiences of people with SSc with a qualitative approach. Problems with swallowing/gastrointestinal symptoms, pain, fatigue and emotional distress as well as problems with household activities and impaired work have been described by Because of the small sample size and the focus on the qualitative analysis in this study, no comparative statistics were calculated. Disease duration refers to the period of time since the fi rst non-Raynaud's manifestation. SSc, systemic sclerosis. Continued patients with SSc in earlier studies, consequences also found in all four countries involved in our study. Personal factors like struggle to master one's life were expressed in different ways in the countries in our study. [bib_ref] Become your own advocate: advice from women living with scleroderma, Mendelson [/bib_ref] for example, also found that taking control of one's illness was a necessary component to maintain a high quality of life. Most concepts were considered by the researchers to be body functions and structures followed by environmental factors. Importantly, similar concepts were derived in different countries; fewer than one-third were found in one country only. The categories found in one country only with the highest percentage (more than one-third of these) were linked to the ICF component environmental factors. This may relate to different healthcare and social security systems, different policies for accessibility to services, but also issues such as public transportation. Nevertheless, people with SSc in Europe may experience rather similar problems and may have similar values-even though countries from east, north and central Europe were involved in this study-while contextual factors may be more different when compared with other continents. We expected to fi nd differences in the environmental factors due to the different situations people are facing; for example, Austria is a conservative corporatist welfare state [bib_ref] Social policy in Europe: its impact on families and work, Den Dulk [/bib_ref] with the traditional values of a patriarchal society, compared with, for example, a Scandinavian welfare regime in Sweden with higher rates of part-time employment among women. [bib_ref] Welfare state regimes, unemployment and health: a comparative study of the relationship..., Bambra [/bib_ref] However, we did not expect to fi nd such considerable differences in the area of body functions and structures. This may be due to cultural values that affect the topics mentioned in a focus group as, for example, affected sex life was only mentioned in Sweden, but not in any other country. While negative and positive aspects were described by the participants in body functions and structures and environmental and personal factors, the concepts in the area of activities and participation related only to negative aspects and dysfunction. In a focus-group study of hand osteoarthritis, just one positive aspect in the area of activities and participation was described, which was the involvement in sports experienced as helpful by the participants, [bib_ref] Patient perspective of hand osteoarthritis in relation to concepts covered by instruments..., Stamm [/bib_ref] while in a similar study in people with systemic lupus erythematosus also only negative issues were identifi ed in the area of activities and participation. [bib_ref] Concepts important to persons with systemic lupus erythematosus and their coverage by..., Stamm [/bib_ref] In the systemic lupus erythematosus study which was conducted only in Austria, a similar number of concepts were identifi ed (92 in total, 28 of these related to body functions and structures, 24 to activities and participation, 25 to environmental factors, 2 to the health condition itself, 6 to personal factors and 7 could not be linked to the ICF). This may also be a specifi c factor of the ICF because positive experiences such as being strong owing to having mastered the disease were linked to personal factors rather than activities and participation according to rules for 'translating' concepts into ICF 'language'. Therefore, it may be important to include resources and facilitators, in addition to the usual problems and barriers, in patient-reported outcome measures and instruments. From this perspective, multidimensional scales may be desirable. The concepts of our study may also be used to analyse the validity of patient-reported instruments, but also for the development of new instruments or the development of an ICF core set for SSc, which is a list of typical ICF categories affected in a certain condition. [bib_ref] Development of ICF Core Sets for patients with chronic conditions, Cieza [/bib_ref] A limitation of our study was that only people with SSc from Europe participated. We therefore recommend that further studies with a similar design are carried out in other parts of the world-for example, places with a different climate. Furthermore, different organ involvement of patients might have infl uenced which ICF categories were identifi ed in the area of body functions and structures. However, in the area of activities and participation and personal factors, the participants of this study identifi ed similar issues consistently in at least two or three different countries. A limitation of group interviews may be that they do not allow for in-depth descriptions from individual participants and thus may limit the richness of the data. This may also be related to the presence of two researchers (one moderator and one assistant) during the group discussion. Most participants in this study were women, which we expected because the prevalence of SSc has an overall female:male ratio of about 3:1. Gender-specifi c differences were explicitly raised in the focus groups in Austria and Romania, but interestingly, were not mentioned in the other two countries. An example was the following description from a participant: "We are also women and each of us cares, more or less, about our looks. Then it is a major inconvenience that we are no longer in control of our hands." However, concerns about changing the appearance of one's body may be also related to the concept 'body image and appearance, including weight maintenance/loss', which emerged in Austria, Romania and Sweden, but not in Switzerland. Furthermore, women from the countries in which no gender-specifi c differences were raised might have considered that appearance is not gender-specifi c. Concerns with physical appearance have also been reported in other qualitative studies, suggesting that it might be a risk of stigmatisation. 16 # Conclusion Twenty-two per cent of all concepts were identifi ed in all four countries. In routine clinical care, these concepts could be used to guide assessment of patients with SSc from a comprehensive bio-psycho-social perspective. In rheumatological rehabilitation and interdisciplinary team care, these aspects could be seen as core aspects and consequences of the disease to be addressed in every patient. [table] Table 1: Demographic data of the participants in each country [/table] [table] Table 2: Concepts mainly related to the ICF components Applying self-treatment strategies, such as movement, exercise, other strategies to keep hands warm, higher temperature at home, being occupied, drinking during eating helps, stopping to smoke, going to a spa, special shampoo against hair loss, diet [/table]

CRISPR interference: a structural perspective CRISPR (cluster of regularly interspaced palindromic repeats) is a prokaryotic adaptive defence system, providing immunity against mobile genetic elements such as viruses. Genomically encoded crRNA (CRISPR RNA) is used by Cas (CRISPR-associated) proteins to target and subsequently degrade nucleic acids of invading entities in a sequence-dependent manner. The process is known as 'interference'. In the present review we cover recent progress on the structural biology of the CRISPR/Cas system, focusing on the Cas proteins and complexes that catalyse crRNA biogenesis and interference. Structural studies have helped in the elucidation of key mechanisms, including the recognition and cleavage of crRNA by the Cas6 and Cas5 proteins, where remarkable diversity at the level of both substrate recognition and catalysis has become apparent. The RNA-binding RAMP (repeat-associated mysterious protein) domain is present in the Cas5, Cas6, Cas7 and Cmr3 protein families and RAMP-like domains are found in Cas2 and Cas10. Structural analysis has also revealed an evolutionary link between the small subunits of the type I and type III-B interference complexes. Future studies of the interference complexes and their constituent components will transform our understanding of the system. # Introduction CRISPRs (cluster of regularly interspaced palindromic repeats) are a prokaryotic defence mechanism against viral infection and horizontal gene transfer. CRISPRs are the largest family of prokaryotic repeats [bib_ref] Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements, Mojica [/bib_ref] and have been found in 48 % of bacterial and 84 % of archaeal sequenced genomes to date . A CRISPR array consists of a series of short identical repeat sequences separated by similarly short variable sequences known as spacers . Located adjacent to the CRISPR array are clusters of cas (CRISPR-associated) genes [4] that encode for the proteins responsible for mediating the CRISPR response to foreign nucleic acids. The spacers are derived from foreign nucleic acids, such as viruses and conjugative plasmids, and provide the host with a 'genetic memory' of threats previously encountered [bib_ref] Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements, Mojica [/bib_ref] [bib_ref] CRISPR elements in Yersinia pestis acquire new repeats by preferential uptake of..., Pourcel [/bib_ref] [bib_ref] Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin, Bolotin [/bib_ref]. New spacers are captured in a poorly understood process known as 'adaptation' and incorporated into the CRISPR locus [bib_ref] CRISPR provides acquired resistance against viruses in prokaryotes, Barrangou [/bib_ref]. The spacers are used to target foreign nucleic acids containing sequences complementary to the spacer, termed protospacers, for degradation [bib_ref] Small CRISPR RNAs guide antiviral defense in prokaryotes, Brouns [/bib_ref] ; the process is termed 'interference'. The first step in the interference pathway is the transcription of the CRISPR array from a promoter located in the 'leader' sequence, an AT-rich region located upstream of the CRISPR array [bib_ref] Identification and characterization of E. coli CRISPR-cas promoters and their silencing by..., Pul [/bib_ref]. The array transcript {pre-crRNA [precursor crRNA (CRISPR RNA)]} is then processed into short crRNAs containing a spacer and flanking repeat fragments [bib_ref] Identification of novel non-coding RNAs as potential antisense regulators in the archaeon..., Tang [/bib_ref]. These crRNAs are subsequently bound by complexes of Cas proteins and used to target homologous foreign dsDNA (doublestranded DNA) or ssRNA (single-stranded RNA) for nucleolytic degradation during CRISPR interference [bib_ref] Small CRISPR RNAs guide antiviral defense in prokaryotes, Brouns [/bib_ref] [bib_ref] RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex, Hale [/bib_ref]. The CRISPR/Cas systems are divided into three main types (I, II and III) on the basis of the identity and organisation of genes within a cas locus [bib_ref] Evolution and classification of the CRISPR-Cas systems, Makarova [/bib_ref]. These types are further divided into a total of ten subtypes (I-A, I-B and so on), each of which expresses a different protein complex responsible for interference [fig_ref] Figure 2: The CRISPR/Cas systems and their respective proteins [/fig_ref]. The Cascade (CRISPR-associated complex for antiviral defence) is the effector complex for type I systems [bib_ref] Small CRISPR RNAs guide antiviral defense in prokaryotes, Brouns [/bib_ref] [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref]. This name was originally used solely for the type I-E complex [bib_ref] Small CRISPR RNAs guide antiviral defense in prokaryotes, Brouns [/bib_ref] , which we here call eCascade, but increasingly Cascade is used more as a general term for all type I complexes. Type II systems use a single protein for interference (Cas9) [bib_ref] A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Jinek [/bib_ref] , whereas the III-B subtype uses the CMR complex [bib_ref] RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex, Hale [/bib_ref]. The interference complex of the III-A subtype has yet to be characterized biochemically, but the similarity of the III-A and III-B operons suggests that interference is indeed mediated by an effector complex rather than a single protein. As a result the putative complex has been termed the CSM complex [bib_ref] Evolution and classification of the CRISPR-Cas systems, Makarova [/bib_ref]. Every CRISPR/Cas system apart from the III-B subtype is thought to target dsDNA by forming an R-loop structure, consisting of a heteroduplex between crRNA and the complementary protospacer strand and a ssDNA (single-stranded DNA) non-complementary strand, followed by degradation by the interference nuclease [bib_ref] Small CRISPR RNAs guide antiviral defense in prokaryotes, Brouns [/bib_ref] [bib_ref] The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA, Garneau [/bib_ref] [bib_ref] CRISPR interference limits horizontal gene transfer in Staphylococci by targeting DNA, Marraffini [/bib_ref] [bib_ref] Tuning in to interference: R-loops and Cascade complexes in CRISPR immunity, Ivancic-Bace [/bib_ref]. The CMR complex targets ssRNA by forming an RNA duplex, which is subsequently cleaved [bib_ref] RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex, Hale [/bib_ref] [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref]. The mechanisms of adaptation and CRISPR interference have been extensively reviewed (see references [bib_ref] CRISPR/Cas system and its role in phage-bacteria interactions, Deveau [/bib_ref] [bib_ref] Memory of viral infections by CRISPR-Cas adaptive immune systems: acquisition of new..., Fineran [/bib_ref] [bib_ref] CRISPR/Cas, the immune system of bacteria and archaea, Horvath [/bib_ref] [bib_ref] CRISPR interference: RNA-directed adaptive immunity in bacteria and archaea, Marraffini [/bib_ref] [bib_ref] CRISPR-based adaptive and heritable immunity in prokaryotes, Van Der Oost [/bib_ref] [bib_ref] RNA-guided genetic silencing systems in bacteria and archaea, Wiedenheft [/bib_ref]. In the present review we will focus on the structural biology of the CRISPR system. Crystal structures are available for eight of the 'core' Cas proteins (those found in multiple subtypes) as well as a number of subtype-specific proteins [fig_ref] Figure 2: The CRISPR/Cas systems and their respective proteins [/fig_ref] and Supplementary Table ## Figure 1 schematic representation of crrna biogenesis and crispr interference Processing events involving nucleic acids are coloured; repeats (black), spacers (red-green) and tracrRNA (magenta). For clarity, a single spacer (red) was used to illustrate the processes, although in actual systems all spacers are processed. Targets are shown in other red shades (lighter for the complementary strand and darker for the non-complementary). The PAMs are shown in blue. The pre-crRNA and interference nucleases are indicated along with the interference complexes. S1 at http://www.biochemj.org/bj/453/bj4530155add.htm). The structures of proteins involved in spacer acquisition have provided interesting insights into their function within the CRISPR/Cas system as well as to similarities to non-Cas proteins, such as the parallels between Cas2 and VapD of the toxin/antitoxin system [bib_ref] Structural and biochemical characterization of HP0315 from Helicobacter pylori as a VapD..., Kwon [/bib_ref] , but will not be discussed further in the present review. EM (electron microscopy) images and structures have been determined for five interference complexes, providing invaluable information on the function of each subunit. CRISPR systems are remarkably diverse and subject to rapid evolutionary change. Analysis of the key structural features of Cas proteins involved in crRNA biogenesis and interference highlights recurring themes and points to evolutionary relationships between apparently distinct protein families. ## Pre-crrna processing and crrna biogenesis crRNA provides the CRISPR/Cas system with the sequence specificity needed to selectively target foreign nucleic acids. Mature crRNAs are produced from a single long transcript of the CRISPR array (pre-crRNA), which is processed to yield spacers with 5 and/or 3 repeat fragments [bib_ref] Identification of novel non-coding RNAs as potential antisense regulators in the archaeon..., Tang [/bib_ref] [bib_ref] Identification of 86 candidates for small non-messenger RNAs from the archaeon Archaeoglobus..., Tang [/bib_ref] [bib_ref] CRISPR families of the crenarchaeal genus Sulfolobus: bidirectional transcription and dynamic properties, Lillestol [/bib_ref]. The method and nature of pre-crRNA processing is dependent on the CRISPR/Cas system. Type I and III systems use the Cas6 endonuclease to cleave pre-crRNA within the repeat sequence [bib_ref] Small CRISPR RNAs guide antiviral defense in prokaryotes, Brouns [/bib_ref] [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref] , with the exception of I-C systems that instead use a catalytic variant of Cas5 [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Cas5d processes pre-crRNA and is a member of a larger family of..., Garside [/bib_ref]. The crRNAs from various type III systems are further processed to reduce or remove the repeat sequence at the 3 end [bib_ref] RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex, Hale [/bib_ref] [bib_ref] Mature clustered, regularly interspaced, short palindromic repeats RNA (crRNA) length is measured..., Hatoum-Aslan [/bib_ref]. The enzyme responsible for this degradation is not yet known. The type II system uses a very different mechanism, requiring the transcript of an anti-sense near-perfect repeat and flanking sequences [tracrRNA (transactivating crRNA)] located adjacent to the CRISPR array for processing [bib_ref] CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III, Deltcheva [/bib_ref]. The duplex formed by pre-crRNA and tracrRNA is bound by Cas9 and cleaved in the repeat sequence by cellular RNase III and then in the spacer by an unknown nuclease to leave a spacer fragment and a 3 repeat fragment [bib_ref] CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III, Deltcheva [/bib_ref]. Cas6 and the catalytic type I-C Cas5 [Cas5c, also confusingly known as Cas5d (Dvulg subtype)] catalyse the same reaction. The pre-crRNA is cleaved upstream of the spacer (8 nt for Cas6, 11 nt for Cas5c) [bib_ref] Small CRISPR RNAs guide antiviral defense in prokaryotes, Brouns [/bib_ref] [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Sequence-and structure-specific RNA processing by a CRISPR endonuclease, Haurwitz [/bib_ref] [bib_ref] Characterization of CRISPR RNA processing in Clostridium thermocellum and Methanococcus maripaludis, Richter [/bib_ref] generating crRNA with a 5 repeatderived sequence known as the '5 -handle' or '5 -tag' that is critical for interference [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref] [bib_ref] Self versus non-self discrimination during CRISPR RNA-directed immunity, Marraffini [/bib_ref]. CRISPR repeats can be divided into twelve families based on sequence and secondary structure [bib_ref] Evolutionary conservation of sequence and secondary structures in CRISPR repeats, Kunin [/bib_ref]. Cas5c targets repeats containing hairpin structures, whereas the subfamilies of Cas6 proteins, which broadly align with the CRISPR/Cas system with which they are associated, can cleave either unstructured or hairpin-containing repeats [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Sequence-and structure-specific RNA processing by a CRISPR endonuclease, Haurwitz [/bib_ref] [bib_ref] An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease..., Sashital [/bib_ref] [bib_ref] Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage, Wang [/bib_ref]. In type I systems, Cas6 can form an integral part of Cascade or it can exhibit a more transient interaction. Cas6e and Cas6f remain tightly bound to their cleaved products with low or subnanomolar affinities, and form part of their respective Cascades [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref] [bib_ref] An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease..., Sashital [/bib_ref] [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref] [bib_ref] Mechanism of substrate selection by a highly specific CRISPR endoribonuclease, Sternberg [/bib_ref]. In fact, the type I-F complex (f Cascade) assembles specifically around a pre-formed Cas6f/crRNA complex [bib_ref] Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR..., Haurwitz [/bib_ref]. Cas6 interacts more transiently with the I-A archaeal Cascade (aCascade) [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] [bib_ref] Characterization of the CRISPR/Cas subtype I-A system of the hyperthermophilic crenarchaeon Thermoproteus..., Plagens [/bib_ref]. Cas6 is not part of the type III-B CMR complex [bib_ref] RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex, Hale [/bib_ref] [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref] , and the associations of Cas6 with the type I-B, I-D and III-A complexes are unclear. ## The structures of cas5c and cas6 Cas5 and Cas6 both belong to the RAMP (repeat-associated mysterious protein) superfamily. These proteins contain one or more RAMP domains, which form ferredoxin-like folds similar to that of the RRM (RNA recognition motif) domain [bib_ref] The mysterious RAMP proteins and their roles in small RNA-based immunity, Wang [/bib_ref] , consisting of a four-stranded antiparallel β-sheet (arranged as β 4 β 1 β 3 β 2 ) flanked on one face by two α-helices located after β 1 and β 3 in a βαββαβ fold [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref]. Five conserved sequence motifs have Typical gene identities are shown for CRISPR/Cas subtypes according to the recent classification by Makarova et al. [bib_ref] Evolution and classification of the CRISPR-Cas systems, Makarova [/bib_ref]. The genes are ordered by function: interference (left) and adaptation (right). The interference proteins are subdivided into the interference nuclease (left, outlined in black), proteins of the interference complex (middle, boxed in red) and pre-crRNA nucleases (right, although some are integral subunits of the interference complexes). The genes are coloured according to conserved domain and protein folds: catalytic RAMPs are shown in blue, non-catalytic RAMPs in light blue, HD nuclease domains in light green, Cas3 helicase domains in dark green, the large subunits in various shades of purple and the small subunits in yellow. Subtypes I-D and II-B are not shown as there is no directly relevant structural data. EM images and structures of the interference complexes (or subcomplex for I-A) are adapted from references 1 [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] , 2 [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] , 3 EMD-5314, 4 [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref] and 5 [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref]. been detected in the superfamily; as yet no single protein has been found to contain all five [bib_ref] A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted..., Makarova [/bib_ref]. Cas6 proteins typically contain two sequential RAMP domains with the glycine-rich loop (motif V of the RAMP superfamily sequence motifs) located between α 2 and β 4 of the second (C-terminal) domain (the prime denotes a structural element in the second domain) [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref] [bib_ref] Crystal structure of hypothetical protein TTHB192 from Thermus thermophilus HB8 reveals a..., Ebihara [/bib_ref] [bib_ref] Cas6 is an endoribonuclease that generates guide RNAs for invader defense in..., Carte [/bib_ref] [bib_ref] Crystal structure of a Cas6 paralogous protein from Pyrococcus furiosus, Park [/bib_ref] [bib_ref] The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA..., Wang [/bib_ref] [bib_ref] Recognition and cleavage of a nonstructured CRISPR RNA by its processing endoribonuclease..., Shao [/bib_ref] [bib_ref] Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site..., Reeks [/bib_ref]. This loop often fits the consensus sequence G GXXXXXG G, where is a hydrophobic residue, X is any residue and the variable region contains at least one positively charged residue [bib_ref] A DNA repair system specific for thermophilic Archaea and bacteria predicted by..., Makarova [/bib_ref]. Other than this motif, the Cas6 proteins exhibit minimal sequence similarity. PaCas6f (Pseudomonas aeruginosa Cas6f) is atypical because it contains what is possibly a severely degraded C-terminal RAMP domain [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref] [bib_ref] Sequence-and structure-specific RNA processing by a CRISPR endonuclease, Haurwitz [/bib_ref]. The C-terminal domain contains four short β-strands that, although they are orientated to form a RAMP β-sheet, are not aligned to do so [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref]. The RAMP helices are not present, but the glycine-rich loop (albeit differing from the consensus sequence) is located between the correct β-strands. The Cas6 homologues contain additional secondary structure elements relative to the RAMP elements, but only one feature is fully conserved: a β-hairpin connecting β 2 and β 3 in the C-terminal domain (we denote this the β 2 -β 3 hairpin) that extends beyond the β-sheet. This hairpin is even conserved in the abnormal Cterminal domain of PaCas6f. Cas5c contains an N-terminal RAMP domain and a C-terminal domain consisting of a three-stranded antiparallel β-sheet [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref] [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Cas5d processes pre-crRNA and is a member of a larger family of..., Garside [/bib_ref] [bib_ref] Conservation and variability in the structure and function of the Cas5d endoribonuclease..., Koo [/bib_ref]. The RAMP domain contains a glycinerich loop that does not match the Cas6 consensus sequence. It also contains a β 2 -β 3 hairpin that is joined by another short β-strand to form a β-sheet. In some Cas5c homologues, two helices are inserted into the tip of the hairpin [bib_ref] Conservation and variability in the structure and function of the Cas5d endoribonuclease..., Koo [/bib_ref]. Due to the hairpin and the glycine-rich loop, this RAMP domain is similar to the Cas6 C-terminal domain, although it also exhibits significant similarity to the N-terminal domain of archaeal Cas6 proteins. In Cas5c, α 2 is not located behind β 4 ; instead, the shorter β 4 (in other RAMPs, β 4 is longer or is followed by an extended strand) allows α 2 to run antiparallel to β 1 (compare [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref] with [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref]. This atypical arrangement could correctly position the residues of the active site, which is located at the intersection of α 1 and α 2 at the top of the β-sheet, a location different to that of Cas6 (see below). The β-sheet of the C-terminal domain does not have a RAMP domain arrangement of secondary structure elements. However, β 1 and β 2 form an extended β-hairpin reminiscent of the β 2 -β 3 hairpin of Cas6, although this is the only feature that is potentially RAMPlike. Thus it is not possible to say with certainty whether the C-terminal domain of Cas5c is a highly divergent RAMP domain. ## Rna binding and cleavage Cas5c and Cas6 are both metal-independent ribonucleases that form products with 5 -hydroxyls and 2 ,3 -cyclic phosphates [bib_ref] Cas5d processes pre-crRNA and is a member of a larger family of..., Garside [/bib_ref] [bib_ref] Sequence-and structure-specific RNA processing by a CRISPR endonuclease, Haurwitz [/bib_ref] [bib_ref] Cas6 is an endoribonuclease that generates guide RNAs for invader defense in..., Carte [/bib_ref] [bib_ref] Structural basis for CRISPR RNA-guided DNA recognition by Cascade, Jore [/bib_ref] , indicative of a general acid/base mechanism involving nucleophilic attack by the deprotonated 2 -hydroxyl on the scissile phosphate. The active site of Cas6 is located between α 1 and the glycine-rich loop, although the exact position of the site varies amongst the subfamilies [fig_ref] Figure 4: RNA binding and catalysis by Cas6 and Cas5cThe structures of [/fig_ref]. Remarkably, the catalytic residues also vary between the proteins and none of the residues are conserved in all of the Cas6 subfamilies. Cas6 enzymes from Pyrococcus furiosus (PfuCas6) and Thermus thermophilus (TtCas6) possess a catalytic triad of histidine, tyrosine and lysine residues similar to the RNA-splicing endonuclease [bib_ref] An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease..., Sashital [/bib_ref] [bib_ref] Cas6 is an endoribonuclease that generates guide RNAs for invader defense in..., Carte [/bib_ref] [bib_ref] RNA-splicing endonuclease structure and function, Calvin [/bib_ref] [bib_ref] Recognition and maturation of effector RNAs in a CRISPR interference pathway, Gesner [/bib_ref]. The tyrosine residue has been assigned as the general base and the histidine residue as the general acid, with the lysine residue stabilizing the pentacoordinate phosphate intermediate. PaCas6f, however, uses a catalytic dyad of histidine and serine residues, with the histidine residue acting as the general base and the serine residue holding the ribose ring in the correct conformation [bib_ref] Csy4 relies on an unusual catalytic dyad to position and cleave CRISPR..., Haurwitz [/bib_ref]. Two active Cas6 paralogues from Sulfolobus solfataricus contain neither a general acid nor a general base, instead using conserved positively charged residues to correctly orientate the substrate and stabilize the pentacoordinate phosphate intermediate [bib_ref] Recognition and cleavage of a nonstructured CRISPR RNA by its processing endoribonuclease..., Shao [/bib_ref] [bib_ref] Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site..., Reeks [/bib_ref]. The presence of a catalytic histidine residue in the N-terminal domain had previously been highlighted as a characteristic feature of Cas6s [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref] , but it is now clear that this is not necessarily the case. The location of the Cas5c active site is different to that of Cas6, suggesting that the active sites evolved independently of each other. The catalytic triad of BhCas5c (Bacillus halodurans Cas5c) consists of a tyrosine residue located in α 1 and histidine and lysine residues in α 2 , similar to the PfuCas6 and TtCas6e active sites [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref]. The lysine is the only residue of the triad that is invariant across the family; the tyrosine residue can be exchanged for histidine (as in the active Cas5c nucleases from Mannheimia succiniciproducens and Xanthomonas oryzae [bib_ref] Cas5d processes pre-crRNA and is a member of a larger family of..., Garside [/bib_ref] [bib_ref] Conservation and variability in the structure and function of the Cas5d endoribonuclease..., Koo [/bib_ref] , phenylalanine or leucine, whereas the catalytic histidine residue can be replaced by other aromatic residues (phenylalanine/tyrosine) (Supplementary Figure S1 at http://www.biochemj. org/bj/453/bj4530155add.htm), but the roles of the residues are not yet understood. None of these supposed catalytic residues are conserved in other Cas5 proteins, perhaps unsurprisingly since only Cas5c is catalytically active. As expected for nucleases that process RNA substrates with a range of secondary structures, multiple modes of RNA binding have been observed across the Cas6 family. This perhaps underlies the variation in the position of the active site as the different modes alter the position of the scissile bond. PfuCas6 and its inactive homologue from Pyrococcus horikoshii (PhCas6nc) bind unstructured RNA in a 'wrap-around' mechanism where the RNA binds in the cleft between the two domains [fig_ref] Figure 4: RNA binding and catalysis by Cas6 and Cas5cThe structures of [/fig_ref] [bib_ref] Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage, Wang [/bib_ref] [bib_ref] The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA..., Wang [/bib_ref]. These enzymes bind the 5 end of the repeat in the cleft between the β-sheets of the two domains and this interaction with the first ∼ 10 nt appears to be the predominant determinant of binding affinity. Although the 3 end of the substrate, including the scissile phosphate, is disordered in the crystal structures, it is predicted to follow the positively charged cleft into the active site [bib_ref] Interaction of the Cas6 riboendonuclease with CRISPR RNAs: recognition and cleavage, Wang [/bib_ref]. TtCas6e, PaCaf6f and a homologue from S. solfataricus (SsoCas6) bind hairpin RNA with the majority of the contacts formed by the C-terminal domain [fig_ref] Figure 4: RNA binding and catalysis by Cas6 and Cas5cThe structures of [/fig_ref]. TtCas6e and SsoCas6 bind the hairpin across the helical face of the protein using a series of basic residues to bind the phosphate backbone of the 3 strand of the hairpin [bib_ref] An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease..., Sashital [/bib_ref] [bib_ref] Recognition and cleavage of a nonstructured CRISPR RNA by its processing endoribonuclease..., Shao [/bib_ref] [bib_ref] Recognition and maturation of effector RNAs in a CRISPR interference pathway, Gesner [/bib_ref]. The RNA hairpin of SsoCas6 is shorter than that of TtCas6e by 3 bp and is predicted to be unstable in solution [bib_ref] Evolutionary conservation of sequence and secondary structures in CRISPR repeats, Kunin [/bib_ref] , meaning that SsoCas6 specifically stabilizes the hairpin conformation. PaCas6f, which shares few Cterminal secondary structure elements with other Cas6 proteins, binds the RNA hairpin between the RAMP β-strands and a helixloop-helix motif, using the first helix to bind the major groove of the RNA [bib_ref] Sequence-and structure-specific RNA processing by a CRISPR endonuclease, Haurwitz [/bib_ref]. In all three of these proteins, the β 2 -β 3 hairpin is inserted into the base of the RNA hairpin, serving to position the scissile phosphate within the active site and, in the case of PaCas6f and SsoCas6, provides key catalytic residues. It seems likely that the β 2 -β 3 hairpin plays a conserved role across the Cas6 family. The method of substrate binding in Cas5c must be significantly different to that observed in Cas6 proteins, because the active sites of the two families are in different locations [fig_ref] Figure 4: RNA binding and catalysis by Cas6 and Cas5cThe structures of [/fig_ref]. In Cas5c, RNA is expected to bind to the helical face of the protein, which in all structures is positively charged, particularly adjacent to the active site [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Cas5d processes pre-crRNA and is a member of a larger family of..., Garside [/bib_ref]. Both domains of Cas5c are implicated in binding the substrate, including the β-sheet encompassing the putative β 2 -β 3 hairpin [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Cas5d processes pre-crRNA and is a member of a larger family of..., Garside [/bib_ref]. However, neither the β 2 -β 3 nor the β 2 -β 3 hairpin can function by inserting at the base of the RNA hairpin, as this would place the scissile phosphate too far away from the active site. A complex structure of Cas5c and substrate is required to determine the exact mode of binding. The method of RNA binding for Cas5c and Cas6 differs from typical RRMs, which contain the same ferredoxin-like fold as RAMPs. Typical RRMs possess two conserved sequence motifs located in β 1 and β 3 (termed RNP2 and RNP1 respectively) that are not present in RAMPs (Supplementary [fig_ref] Figure 2: The CRISPR/Cas systems and their respective proteins [/fig_ref] at http://www.biochemj.org/bj/453/bj4530155add.htm) [bib_ref] The RNA recognition motif, a plastic RNA-binding platform to regulate post-transcriptional gene..., Maris [/bib_ref] [bib_ref] RNA recognition motifs: boring? Not quite, Clery [/bib_ref]. These motifs allow RRMs to bind ssRNA or ssDNA across the face of the β-sheet [bib_ref] Crystal-structure at 1.92 angstrom resolution of the RNA-binding domain of the U1A..., Oubridge [/bib_ref] [bib_ref] Crystal structure of the two-RRM domain of hnRNP A1 (UP1) complexed with..., Ding [/bib_ref] , although not hairpin or dsRNA (double-stranded RNA), whereas RAMPs bind ssRNA or hairpin RNA through diverse modes of binding. The active sites appear to have evolved independently for Cas6 and Cas5c, and even within the Cas6 family there is no universally conserved catalytic mechanism. Given that the catalytic rate constants of these enzymes, at 1-5 min − 1 [bib_ref] An RNA-induced conformational change required for CRISPR RNA cleavage by the endoribonuclease..., Sashital [/bib_ref] [bib_ref] Mechanism of substrate selection by a highly specific CRISPR endoribonuclease, Sternberg [/bib_ref] , are of the same order as those observed for catalytic RNA [bib_ref] The origins of RNA catalysis in ribozymes, Lilley [/bib_ref] , these enzymes may be more constrained by the need to recognize pre-crRNA specifically than by a requirement to turn over rapidly. ## The proteins of the interference complexes Atomic level detail structures are now available for a number of individual proteins that are involved in interference. In addition, EM structures have been solved for a number of the interference complexes [fig_ref] Figure 2: The CRISPR/Cas systems and their respective proteins [/fig_ref]. The highest resolution structures available are those of the Escherichia coli eCascade in complex with crRNA and with a crRNA/protospacer RNA duplex at resolutions of 8 and 9 Å (1 Å = 0.1 nm) respectively [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. Lower resolution images and structures are also available for the B. halodurans cCascade [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] , Ps. aeruginosa f Cascade [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref] and S. solfataricus CMR complex [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref] as well as the core complex of S. solfataricus aCascade [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref]. Although the overall complex topologies can be discerned, the resolution of these structures has precluded reliable placement of individual proteins within the complex. ## Cas7, the backbone of the type i complex The structural backbone of Cascade is composed of multiple monomers of Cas7 [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. In eCascade, Cas7 assembles into a helical hexameric structure with crRNA binding in a groove formed along the outer face of the oligomer [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. This helical arrangement is conserved in the core complex of the S. solfataricus aCascade, although this complex of Cas5 and Cas7 forms oligomers of variable length [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref]. It is possible that further factors are needed to produce a complex of defined length or perhaps aCascade exhibits greater structural plasticity than eCascade. A similar helical arrangement to eCascade was observed in EM images of cCascade [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] , and, although it was not possible to unambiguously define the quaternary structure of the complex, it is probable that the six Cas7 subunits of the complex form the same backbone. f Cascade contains six Csy3 subunits with a similar twisted topology to both cCascade and eCascade [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref]. This, combined with secondary structure predictions and MS fragmentation analysis, has recently led to the hypothesis that Csy3 actually belongs in an expanded Cas7 family [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref] [bib_ref] Native tandem and ion mobility mass spectrometry highlight structural and modular similarities..., Van Duijn [/bib_ref]. Similar structure predictions place Csc2 of dCascade in the Cas7 family [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref] , suggesting that the Cas7 helical backbone is a conserved and perhaps characteristic feature of all Cascade complexes. The structure of Cas7 from one of the S. solfataricus aCascade complexes [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] (termed SsoCas7) contains a central RAMP fold modified with an additional αβα motif located immediately after β 4 [fig_ref] Figure 5: The structure of Cas7, the core subunit of Cascade [/fig_ref]. This motif adds a fifth strand to the β-sheet (β 5 β 4 β 1 β 3 β 2 ) with the two helices on either side of β 5 . The loop between α 2 and β 4 is disordered in the structure and is not glycinerich, a conserved feature of the Cas7 family [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref]. Significant insertions are located between each of the four β-strands; these form two distinct regions above and below the β-sheet to form a crescent-shaped molecule [fig_ref] Figure 5: The structure of Cas7, the core subunit of Cascade [/fig_ref]. Residues located in the cleft of SsoCas7 have been implicated in binding crRNA [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref]. The structure of eCascade shows that the E. coli Cas7 adopts a similar topology to SsoCas7 and that the cleft forms the extended groove along the helical assembly of Cas7 [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. Given the likely ubiquitous nature of the Cas7 backbone, it is probable that all Cascade complexes bind crRNA in the same manner. ## Non-catalytic variants of cas5 Although Cas5c possesses catalytic activity, the other members of the Cas5 family are non-catalytic and are limited to structural roles. In both aCascade and eCascade, Cas5 interacts stably with Cas7 [bib_ref] Structural and functional characterization of an archaeal clustered regularly interspaced short palindromic..., Lintner [/bib_ref] [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. Cas5e also interacts with Cse1 and Cse2 in eCascade and appears to help stabilize the protospacer-bound conformation of the complex [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. cCascade contains two copies of Cas5c, which appear to occupy the positions of Cas5 and Cas6e in eCascade [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. Cas5c from Streptococcus pyogenes and X. oryzae bind dsDNA, which could be mimicking target dsDNA or the heteroduplex of the interference R-loop [bib_ref] Conservation and variability in the structure and function of the Cas5d endoribonuclease..., Koo [/bib_ref]. Therefore Cas5c seems to be able to function as both a catalytic Cas6 equivalent and a structural Cas5 equivalent. Of the Cascade complexes, only dCascade and f Cascade do not contain Cas5 [bib_ref] Evolution and classification of the CRISPR-Cas systems, Makarova [/bib_ref]. On the basis of secondary structure predictions, Makarova et al. [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref] predicted that Csc1 (I-D) and Csy2 (I-F) belong to the Cas5 family. EM images and the small-angle X-ray scattering (SAXS) structure of f Cascade place Csy2 in a similar position to the structural Cas5s of cCascade and eCascade [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref] [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref]. However, the fragmentation patterns of eCascade and f Cascade suggest that Csy2 does not interact with Csy3 (probable Cas7 equivalent) in the same manner as Cas5 and Cas7 from eCascade, leading van Duijn et al. [bib_ref] Native tandem and ion mobility mass spectrometry highlight structural and modular similarities..., Van Duijn [/bib_ref] to conclude that f Cascade does not contain a Cas5 equivalent. Further data are required to settle the relationships between the complexes. ## The small subunits of the interference complexes Several of the interference complexes contain so-called 'small' subunits, which are typically <200 residues. These proteins are Csa5 (I-A), Cse2 (I-E), Csm2 (III-A) and Cmr5 (III-B) and it has been hypothesized that these proteins belong to a single family (Cas11) [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref]. Analysis of the structures of Csa5 [bib_ref] Structure of the archaeal Cascade subunit Csa5: relating the small subunits of..., Reeks [/bib_ref] , Cse2 although structural homology can be detected, the evolutionary links between the proteins are complex. Cse2 contains N-and C-terminal domains that consist of four and five α-helices respectively. The N-terminal domain is homologous with the core structure of Cmr5, whereas the C-terminal domain is homologous with one of the domains of Csa5 [fig_ref] Figure 6: The small subunits of interference complexes Comparison of T [/fig_ref]. Csa5 consists of an α-helical domain (homologous with the Cse2 C-terminal domain) and a β-sheet domain that is not homologous with Cse2 or Cmr5. In fact, this domain is very poorly conserved across the Csa5 family and is likely to vary significantly between homologues. Possible evolutionary scenarios for the homology include fusion of csa5 and cmr5 genes to form cse2 or the evolution of the three proteins from a single cse2-like gene with domain loss to form Csa5 and Cmr5 [bib_ref] Structure of the archaeal Cascade subunit Csa5: relating the small subunits of..., Reeks [/bib_ref]. Csm2, the remaining small subunit for which there is no structure available, may be critical for determining the likely scenario, although it is certainly possible that Csm2 may not possess any homology with the other small subunits. Makarova et al. [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref] suggested that the Cas8 C-terminal domain, which is predicted to be helical, might be homologous with the small subunits, although no experimental structure exists to confirm this. The Cse2 dimer is an integral part of eCascade [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref] and is responsible for stabilizing the R-loop, increasing the affinity of eCascade for dsDNA approximately 10-fold [bib_ref] Cascade-mediated binding and bending of negatively supercoiled DNA, Westra [/bib_ref]. Cse2 alone binds non-specifically to dsDNA and ssRNA [bib_ref] Nucleic acid binding surface and dimer interface revealed by CRISPR-associated CasB protein..., Nam [/bib_ref]. Conversely, the S. solfataricus Csa5 does not stably interact with Cas5/Cas7 in the presence of crRNA or with nucleic acids alone [bib_ref] Structure of the archaeal Cascade subunit Csa5: relating the small subunits of..., Reeks [/bib_ref]. Cmr5, in contrast with both Csa5 and Cse2, appears to be non-essential to the function of the CMR complex [bib_ref] RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex, Hale [/bib_ref]. Thus we conclude that the similarity of the small subunits is structural rather than functional. ## The large subunits of the interference complexes Similarly to the small subunits, each of the type I and III interference complexes contains a 'large' (>500 residues) subunit: Cas8 (I-A, I-B, I-C), Cse1 (I-E), Csy1 (I-F) and Cas10 (I-D, III-A and III-B). Cas10 was originally predicted to be a polymerase (hence the name polymerase cassette for the III-B subtype) on the basis of sequence features typical of a palm domain commonly found in polymerases and cyclases [bib_ref] A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted..., Makarova [/bib_ref]. Subsequently it was proposed that all of the large subunits were homologous and part of a Cas10 superfamily [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref]. However, recent structures of a type III-B Cas10 [bib_ref] Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex, Cocozaki [/bib_ref] [bib_ref] Crystal structure of Cmr2 suggests a nucleotide cyclase-related enzyme in type III..., Zhu [/bib_ref] , denoted Cas10b, show that, although the prediction of the palm domain was correct (albeit more akin to cyclases), no significant structural homology exists with Cse1 [bib_ref] Crystal structure of the largest subunit of a bacterial RNA-guided immune complex..., Mulepati [/bib_ref] [bib_ref] Mechanism of foreign DNA selection in a bacterial adaptive immune system, Sashital [/bib_ref] (PDB codes 4H3T and 4EJ3). This argues against a single common ancestor for all of the large subunits. ## Cas10, the large subunit of type iii systems Cas10 is the defining protein of the type III system and consists of an N-terminal HD (histidine-aspartate) phosphohydrolase domain (for which there is no structure) and a C-terminal region (Cas10 dHD ) that contains the palm domain [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref]. Cas10b dHD from P. furiosus consists of two adenylate cyclase-like domains (denoted D1 and D3) and two α-helical domains (D2 and D4) [fig_ref] Figure 7: The large subunits of interference complexes [/fig_ref] [bib_ref] Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex, Cocozaki [/bib_ref] [bib_ref] Crystal structure of Cmr2 suggests a nucleotide cyclase-related enzyme in type III..., Zhu [/bib_ref]. D2 is not significantly homologous with known structures, but D4 is structurally homologous with Cmr5 and the N-terminal domain of Cse2, although sequence conservation is minimal and the biological implications of the homology are unclear. A typical adenylate cyclase domain consists of a ferredoxin-like fold with a C-terminal α 3 β 5 α 4 β 6 β 7 modification, which creates a seven-stranded β-sheet with the two additional helices located on either side of the sheet [bib_ref] Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c, Sinha [/bib_ref]. D1 and D3 lack some of these key structural elements: D3 lacks α 4 and β 6 , whereas D1 lacks every additional element bar α 3 . Individually, D1 and D3 are most similar to the type III adenylate cyclase from Mycobacterium tuberculosis [bib_ref] Origin of asymmetry in adenylyl cyclases: structures of Mycobacterium tuberculosis Rv1900c, Sinha [/bib_ref]. However, these bacterial cyclases are typically homodimers, whereas D1 and D3 of Cas10b dHD exist as a pseudoheterodimer more similar to the arrangement of mammalian cyclases [bib_ref] The class III adenylyl cyclases: multi-purpose signalling modules, Linder [/bib_ref]. The orientation between D1 and D3 is markedly different to that of typical cyclases which, combined with the loss of key structural and sequence features, is consistent with PfuCas10b dHD lacking a cyclase-like catalytic activity, although D3 retains the ability to bind ADP [bib_ref] Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex, Cocozaki [/bib_ref]. In the CMR complex Cas10b interacts with Cmr3, an interaction observed in both S. solfataricus and P. furiosus [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref] [bib_ref] Structure of the cmr2-cmr3 subcomplex of the cmr RNA silencing complex, Shao [/bib_ref]. The structure of the P. furiosus Cas10b dHD -Cmr3 complex shows that the two proteins form a heterodimer with the interface formed by D1 of Cas10b dHD and one face of Cmr3 (see below) [bib_ref] Structure of the cmr2-cmr3 subcomplex of the cmr RNA silencing complex, Shao [/bib_ref]. At the interface between the two proteins is a highly positively charged cleft ∼ 50 Å in length, which is suggestive of a role in crRNA binding. The nucleotide bound by D3 in both the Cas10b dHD and Cas10b dHD -Cmr3 structures lies at the centre of this cleft and so could be mimicking crRNA binding by the complex rather than substrate binding by the 'cyclase' domains of Cas10b dHD . This is consistent with the nucleotide binding in a different orientation to that observed in cyclases. If the Cas10b-Cmr3 complex does bind to part of the crRNA, the remainder of the crRNA must be bound by other subunits of the CMR complex. Three subunits of the complex (Cmr1, Cmr4 and Cmr6) are RAMPs and thus are plausible candidates. Makarova et al. [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref] have predicted Cmr4 and Cmr6 to be Cas7 homologues. However, EM structures of the CMR complex (which targets ssRNA and not dsDNA) show that it is more compact than Cascade and lacks a central helical structure [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref]. ## Cse1, the pam (protospacer adjacent motif) sensor of ecascade The structures of Cse1 from T. thermophilus [bib_ref] Crystal structure of the largest subunit of a bacterial RNA-guided immune complex..., Mulepati [/bib_ref] [bib_ref] Mechanism of foreign DNA selection in a bacterial adaptive immune system, Sashital [/bib_ref] (PDB code 4EJ3) and Acidimicrobium ferrooxidans (PDB code 4H3T) consist of an N-terminal mixed α/β domain with a novel fold and a C-terminal four-helix bundle [fig_ref] Figure 7: The large subunits of interference complexes [/fig_ref]. In eCascade, Cse1 is responsible for recognition of the PAM, a short (2-5 nt) conserved sequence located immediately next to the protospacer that is required for interference [bib_ref] Short motif sequences determine the targets of the prokaryotic CRISPR defence system, Mojica [/bib_ref]. Cascade recognizes a PAM located 5 to the protospacer [bib_ref] Short motif sequences determine the targets of the prokaryotic CRISPR defence system, Mojica [/bib_ref] and, at least for eCascade, PAM recognition uses the complementary strand [bib_ref] CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled..., Westra [/bib_ref]. Target dsDNA lacking a PAM is bound weakly by eCascade [bib_ref] CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled..., Westra [/bib_ref] [bib_ref] Interference by clustered regularly interspaced short palindromic repeat (CRISPR) RNA is governed..., Semenova [/bib_ref] and is resistant to cleavage [bib_ref] In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus, Sinkunas [/bib_ref] , consistent with the observation that mutations in the PAM can prevent interference [bib_ref] RNA-guided complex from a bacterial immune system enhances target recognition through seed..., Wiedenheft [/bib_ref] [bib_ref] Phage response to CRISPR-encoded resistance in Streptococcus thermophilus, Deveau [/bib_ref]. The N-terminal domain of Cse1 contains a loop (L1, [fig_ref] Figure 7: The large subunits of interference complexes [/fig_ref] that is disordered in all of the available crystal structures, but is critical for PAM recognition [bib_ref] Crystal structure of the largest subunit of a bacterial RNA-guided immune complex..., Mulepati [/bib_ref] [bib_ref] Mechanism of foreign DNA selection in a bacterial adaptive immune system, Sashital [/bib_ref]. Analysis of the eCascade structures led Mulepati et al. [bib_ref] Crystal structure of the largest subunit of a bacterial RNA-guided immune complex..., Mulepati [/bib_ref] and Sashital et al. [bib_ref] Mechanism of foreign DNA selection in a bacterial adaptive immune system, Sashital [/bib_ref] to suggest that L1 binds to the crRNA 5 -handle and PAM in the absence and presence of target DNA respectively. Cse1 is also critical for binding to negatively supercoiled dsDNA, both specifically to a protospacer and also non-specifically, a function that is dependent on the L1 loop [bib_ref] Structural basis for CRISPR RNA-guided DNA recognition by Cascade, Jore [/bib_ref] [bib_ref] Crystal structure of the largest subunit of a bacterial RNA-guided immune complex..., Mulepati [/bib_ref] [bib_ref] Mechanism of foreign DNA selection in a bacterial adaptive immune system, Sashital [/bib_ref]. Sashital et al. [bib_ref] Mechanism of foreign DNA selection in a bacterial adaptive immune system, Sashital [/bib_ref] have proposed that Cse1 scans dsDNA for PAM sequences and once in contact destabilizes the duplex to allow for target recognition, first through a 5 seed sequence and then along the remainder of the target. Other Cascade complexes lack Cse1 and must use a different protein for PAM sensing, although their identities have not been established. Cas8 and Csy1 are candidates as they dissociate easily from their respective complexes (similar to Cse1 and eCascade) and EM images suggest that they are located in a similar position to Cse1 within their complexes [bib_ref] Cas5d protein processes pre-crRNA and assembles into a cascade-like interference complex in..., Nam [/bib_ref] [bib_ref] Structures of the RNA-guided surveillance complex from a bacterial immune system, Wiedenheft [/bib_ref] [bib_ref] Native tandem and ion mobility mass spectrometry highlight structural and modular similarities..., Van Duijn [/bib_ref]. ## Cmr3, a type iii-b cas6-like protein Cmr3 is a RAMP protein of the CMR complex and the structure of PfuCmr3, available only in complex with Cas10b dHD , shows that it contains two RAMP domains arranged in a similar manner to Cas6 (compare [fig_ref] Figure 8: The structure of Cmr3 [/fig_ref] with [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref] [bib_ref] Structure of the cmr2-cmr3 subcomplex of the cmr RNA silencing complex, Shao [/bib_ref]. The C-terminal domain contains two of the conserved features of Cas6: the β 2 -β 3 hairpin and the glycine-rich loop, both of which adopt similar conformations to those seen in Cas6 proteins. The Cmr3 glycine-rich loop also exhibits a similar consensus sequence to that of Cas6 (XXXXXGϕG, where ϕ is an aromatic residue, X is any residue and the variable region contains at least one positively charged residue) (Supplementary [fig_ref] Figure 3: The structures of catalytic RAMP proteins [/fig_ref] at http://www.biochemj.org/bj/453/bj4530155add.htm). In the Nterminal domain, a β-strand located after α 2 forms a β-hairpin with β 4 , as is also seen in the Pyrococcus and Sulfolobus Cas6 homologues [bib_ref] Cas6 is an endoribonuclease that generates guide RNAs for invader defense in..., Carte [/bib_ref] [bib_ref] Crystal structure of a Cas6 paralogous protein from Pyrococcus furiosus, Park [/bib_ref] [bib_ref] The impact of CRISPR repeat sequence on structures of a Cas6 protein-RNA..., Wang [/bib_ref] [bib_ref] Recognition and cleavage of a nonstructured CRISPR RNA by its processing endoribonuclease..., Shao [/bib_ref] [bib_ref] Structure of a dimeric crenarchaeal Cas6 enzyme with an atypical active site..., Reeks [/bib_ref] , with the turn of the hairpin containing the two conserved glycine residues identified by Makarova et al. [bib_ref] Unification of Cas protein families and a simple scenario for the origin..., Makarova [/bib_ref] as an N-terminal glycine-rich loop. The tip of this loop is disordered, but since it is only three residues in length it acts more as a turn rather than the extended loop seen in many RAMPs. Cmr3 exhibits two significant deviations from Cas6. α 2 is replaced by a short β-strand located immediately prior to the Cterminal glycine-rich loop, similar to the β-strand located before the N-terminal glycine-rich loop. The second difference is the presence of a significant structural insertion located between β 2 and β 3 of the N-terminal domain. This insertion consists of two short helices and seven β-strands and packs against the C-terminal β-sheet. The insertion and the β 2 -β 3 hairpin together form the interface with Cas10b dHD and line the putative crRNA-binding cleft. ## The interference nucleases During interference, invading nucleic acids detected by base pairing with crRNA are targeted for degradation by an interference nuclease. In type I systems this is the HD metaldependent nuclease domain of Cas3, which is recruited to Cascade rather than being an integral component [bib_ref] CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled..., Westra [/bib_ref]. Type II systems use Cas9 as the sole interference protein with the HNH-like and RuvClike nuclease domains cleaving the complementary and noncomplementary strands of the R-loop respectively [bib_ref] A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity, Jinek [/bib_ref] [bib_ref] Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria, Gasiunas [/bib_ref]. The interference nucleases of the type III systems are unknown. The nuclease is within the CMR complex, but Cas10b and Cmr5 have been discounted, as has the Sulfolobales-specific protein Cmr7 [bib_ref] RNA-guided RNA cleavage by a CRISPR RNA-Cas protein complex, Hale [/bib_ref] [bib_ref] Structure and mechanism of the CMR complex for CRISPR-mediated antiviral immunity, Zhang [/bib_ref] [bib_ref] Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex, Cocozaki [/bib_ref]. two domains are expressed as separate proteins (Cas3 and Cas3 respectively); other variations are also known, such as domain fusion to other Cas proteins (for example, Cas3-Cas2 in the I-F subtype and Cas3-Cse1 in some I-E systems) and inversion of the domain order [fig_ref] Figure 2: The CRISPR/Cas systems and their respective proteins [/fig_ref] [bib_ref] Evolution and classification of the CRISPR-Cas systems, Makarova [/bib_ref] [bib_ref] A putative RNA-interference-based immune system in prokaryotes: computational analysis of the predicted..., Makarova [/bib_ref] [bib_ref] CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled..., Westra [/bib_ref]. Cas3 is recruited by Cascade after R-loop formation where it catalyses the unwinding and degradation of the invading DNA [bib_ref] CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled..., Westra [/bib_ref] [bib_ref] In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus, Sinkunas [/bib_ref]. Cas3 proteins contain all five HD superfamily sequence motifs (H-HD-H-H-D) and the structures of TtCas3 HD (HD domain of TtCas3) and MjaCas3 (Methanocaldococcus jannaschii Cas3 ) revealed eight conserved helices, five of which are characteristic of the HD superfamily [fig_ref] Figure 9: The structures of Cas protein HD domains [/fig_ref] [bib_ref] Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme..., Beloglazova [/bib_ref] [bib_ref] Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short..., Mulepati [/bib_ref]. In the TtCas3 HD structure a single Ni 2 + ion is bound by motifs I, II and V (site 1), whereas site 2 (a binding site formed by motifs II, III and IV) remains unoccupied [fig_ref] Figure 9: The structures of Cas protein HD domains [/fig_ref]. Metal binding at site 2 has been observed in a number of HD domains (for example, see PDB codes 2OGI, 2O08, 2PQ7, 3CCG and 3HC1) and its absence in the TtCas3 HD structure is likely to be a crystal artefact. The MjaCas3 structure shows a Ca 2 + ion bound at site 2 as well as a second ion bound by the histidine of motif II (site 3) [fig_ref] Figure 9: The structures of Cas protein HD domains [/fig_ref]. However, the binding at site 3 and the lack of binding at site 1 are likely to be artefacts resulting from the protein engineering required for crystallization. Characterization of type I-E Cas3 nuclease domains from T. thermophilus, Streptococcus thermophilus, and E. coli and the type I-A Cas3 proteins from M. jannaschii and P. furiosus showed that they are all metal-dependent nucleases specific for ssDNA, although the Cas3 proteins also cleave ssRNA in vitro [bib_ref] Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme..., Beloglazova [/bib_ref] [bib_ref] Structural and biochemical analysis of nuclease domain of clustered regularly interspaced short..., Mulepati [/bib_ref] [bib_ref] Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas..., Sinkunas [/bib_ref]. These proteins are both endo-and exo-nucleases, with the latter activity proceeding in the 3 →5 direction. MjaCas3 , SthCas3 (Streptococcus thermophilus Cas3) and EcoCas3 (E. coli Cas3) cleave R-loops, the biological substrate of Cas3 and MjaCas3 and SthCas3 have been shown to target the noncomplementary ssDNA strand specifically [bib_ref] CRISPR immunity relies on the consecutive binding and degradation of negatively supercoiled..., Westra [/bib_ref] [bib_ref] In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus, Sinkunas [/bib_ref] [bib_ref] Structure and activity of the Cas3 HD nuclease MJ0384, an effector enzyme..., Beloglazova [/bib_ref]. Structural data is not available for the helicase domain of Cas3, but the type I-E helicase domains of SthCas3 and EcoCas3 catalyse the 3 →5 Mg 2 + -and ATP-dependent unwinding of dsDNA and DNA/RNA duplexes [bib_ref] Cas3 is a single-stranded DNA nuclease and ATP-dependent helicase in the CRISPR/Cas..., Sinkunas [/bib_ref] [bib_ref] Helicase dissociation and annealing of RNA-DNA hybrids by Escherichia coli Cas3 protein, Howard [/bib_ref]. Nicking of the non-complementary strand by the HD domain followed by the unwinding of the DNA duplex by the helicase domain would allow for progressive degradation [fig_ref] Figure 4: RNA binding and catalysis by Cas6 and Cas5cThe structures of [/fig_ref] at http://www.biochemj.org/bj/453/bj4530155add.htm). The complementary strand is also targeted by Cas3 [bib_ref] In vitro reconstitution of Cascade-mediated CRISPR immunity in Streptococcus thermophilus, Sinkunas [/bib_ref] and would occur after dissociation of DNA from the R-loop. ## The hd domains of cas10 proteins Cas10 proteins contain N-terminal HD domains that are highly divergent from typical HD domains, being both shorter than classical HD proteins and lacking characteristic motifs (Supplementary [fig_ref] Figure 5: The structure of Cas7, the core subunit of Cascade [/fig_ref] at http://www.biochemj.org/bj/453/bj4530155add. htm) [bib_ref] Divergence and convergence in enzyme evolution, Galperin [/bib_ref]. A homology model of Cas10a from S. thermophilus built using PHYRE2 [bib_ref] Protein structure prediction on the Web: a case study using the Phyre..., Kelley [/bib_ref] shows that motifs II, III and IV could co-ordinate a metal ion in a similar way to that of site 2 of Cas3 [fig_ref] Figure 9: The structures of Cas protein HD domains [/fig_ref]. Therefore this domain could also be catalytically active and might potentially act as the interference nuclease of the CSM complex, although so far experimental confirmation is lacking. In contrast, Cas10b only contains motif II and so is unlikely to be an active nuclease, consistent with the observation that the Cas10b HD domain is not necessary for interference by the CMR complex [bib_ref] Structure of the Cmr2 subunit of the CRISPR-Cas RNA silencing complex, Cocozaki [/bib_ref] , perhaps unsurprising since this complex targets RNA. ## Concluding remarks The structural biology of the CRISPR system provides a wealth of information on the evolution and mechanisms of the proteins involved. It has revealed the underlying relationships between highly divergent proteins that are difficult or impossible to detect using bioinformatic approaches (however heroic) alone. The RAMP (or RAMP-like) domains, present in the Cas2, Cas5, Cas6, Cas7, Cas10 and Cmr3 families, are the leitmotif of the system, providing RNA-binding and -cleavage functionalities that are central to the process. The backbone of all type I complexes is likely to be a helical arrangement of Cas7, and a similar arrangement of Cas7-like RAMP subunits may be found in the CSM complex, given that it, too, targets dsDNA. Key challenges for crystallography include the structure of the Cas9 protein of type II systems, which has so far evaded attempts to place it in a wider context. Structures of the large and small subunits of the various type I and type III-A complexes are expected to clarify the relationships between the different families, and we can look forward to some simplification of the overall picture as these relationships become apparent. Finally, atomic level structural information on the ∼ 400 kDa CRISPR interference complexes remains a grand challenge in molecular biology, one that has been taken up enthusiastically by the structural biology community. For clarity, the unbound RNA hairpin is not shown. The two RRM RNA-binding consensus sequences are shown beneath the structure. The RPM domain is coloured in the same manner as the RAMP domain in the main text. [fig] Figure 2: The CRISPR/Cas systems and their respective proteins [/fig] [fig] Figure 3: The structures of catalytic RAMP proteins (A) Topology diagram of a RAMP domain. The β-strands are shown in blue and the α-helices in cyan. The glycine-rich loop found in many RAMPs is shown in yellow and the β 2 -β 3 hairpin observed in some RAMPs is shown in green. The N-and C-termini are shown as blue and red spheres respectively. (B) The structure of TtCas6e (PDB code 1WJ9) highlighting the two RAMP domains that may have arisen from a pseudo-duplication event. Secondary structural elements are labelled as described in the text. Conserved RAMP elements are coloured as in (A) and non-conserved elements in grey. Disordered regions are shown as broken black lines. (C) The atypical C-terminal domain of PaCas6f (PDB code 2XLK) that probably diverged from the standard RAMP fold. The recognizable features are labelled. (D) The structure of BhCas5c (PDB code 4F3M), a catalytic variant of the typically non-catalytic Cas5 family. The short β 4 strand and parallel α 2 helix are boxed in black. The possible β 2 -β 3 hairpin in the C-terminal domain is shown in black. [/fig] [fig] Figure 4: RNA binding and catalysis by Cas6 and Cas5cThe structures of (A) PfuCas6(PDB code 3PKM), (B) SsoCas6 (PDB code 4ILL), (C) TtCas6e (PDB code 2Y8W) and (D) PaCas6f (PDB code 2XLK) in complex with RNA (red). The glycine-rich loop is shown in yellow and the catalytic residues as magenta sticks. (E) The structure of BhCas5c (PDB 4F3M) highlighting the position of the active site (magenta). The four structures are shown to the same scale and same orientation. A three-dimensional representation of this Figure is available at http://www.biochemj.org/bj/453/0155/bj4530155add.htm. [/fig] [fig] Figure 5: The structure of Cas7, the core subunit of Cascade (A) The structure of SsoCas7 (PDB code 3PS0) where the central RAMP domain is extended by an αβα motif (orange) and flanked by two unique domains (grey). The proposed crRNA-binding cleft located across the face of the β-sheet is indicated. (B) Topology diagram of SsoCas7 showing the connectivity of the RAMP fold relative to the other domains. [/fig] [fig] Figure 6: The small subunits of interference complexes Comparison of T. thermophilus Cmr5 (PDB code 2ZOP, left), T. thermophilus Cse2 (PDB code 2ZCA, middle) and S. solfataricus Csa5 (PDB code 3ZC4, right). The N-terminal domain of Cse2 (light orange) is superimposed on Cmr5 (blue) and the C-terminal domain of Cse2 (yellow) is superimposed on Csa5 (green). [/fig] [fig] Figure 7: The large subunits of interference complexes (A) The structure of PfuCas10b dHD (PDB code 3UNG) in complex with ADP (red sticks). The ferredoxin-like folds are coloured as for RAMPs and the additional adenylate cyclase elements are shown in orange. D4 is shown in yellow to highlight its homology with the small subunits. The three metal ions are shown as grey spheres. Inset: schematic diagram showing the relative positions of the four domains (D1-D4) with the cyclase-like domains in blue and the small subunit-like domain in yellow. (B) The structure of the Cas10b dHD -Cmr3 complex (PDB code 4H4K) with Cmr3 shown in navy blue and Cas10b dHD as in (A). The putative crRNA-binding cleft is indicated with a solid black line. (C) The structure of Cse1 from T. thermophilus (PDB code 4AN8) with the disordered loop L1 indicated. [/fig] [fig] Figure 8: The structure of Cmr3 (A) The structure of PfuCmr3 (PDB code 4H4K) showing the RAMP elements and the structural insertion in the N-terminal domain (orange). (B) Topology diagram of PfuCmr3 highlighting the conserved RAMP features and the connectivity of the insertion domain. [/fig] [fig] Figure 9: The structures of Cas protein HD domains (A) The structure of TtCas3 HD (PDB code 3SKD) with the conserved HD superfamily helices in green and numbered. The Ni 2 + ion is shown as a dark grey sphere. Residues 222-260 are not shown as they are predicted to belong to the helicase domain. (B) A homology model of the HD domain of Cas10a from S. thermophilus created using PHYRE2 and consisting of residues 4-79. The four HD domain helices are coloured in green and labelled. (C-E) Views of the active sites of (C) TtCas3 HD , (D) MjaCas3 (PDB code 3S4L) and (E) SthCas10a HD . The HD superfamily motifs are shown as sticks with motif numbers in parentheses and the metal ions as grey spheres with site numbers in white. [/fig] [fig] c: The Authors Journal ompilation 2013 Biohemial Soiety of the non-omplementary strand (Supplementary [/fig] [fig] Figure S2: The structure of U1A spliceosomal protein, a typical RRM protein, in complex with RNA (red) (PDB code 1URN) [/fig] [table] Table S1: Details of all of the crystal structures of Cas proteins available at the time of writing [/table]

Saved by the Scan: A Case of Early Detection of Sarcomatoid Mesothelioma # Introduction Malignant pleural mesothelioma (MPM) is an aggressive tumor originating from mesothelial cells lining the lung pleura, peritoneum, tunica vaginalis, and pericardium, listed in descending order of occurrence. The incidence of MPM in the United States in 2018 was 0.7 per 100,000 people. The three main histological subtypes of MPM include epithelioid, sarcomatoid, and biphasic, with sarcomatoid being the least common, accounting for 10% of all cases, and the most aggressive type. There is also a subtype of sarcomatoid mesothelioma called desmoid sarcomesothelioma, which is the focus of this report. The occurrence of MPM is attributed to mineral fiber exposure such as asbestos, and the median duration of time to develop mesothelioma following asbestos exposure is 32 years [bib_ref] Peritoneal mesothelioma: a review, Bridda [/bib_ref] [bib_ref] Clinical diagnosis of malignant pleural mesothelioma, Bianco [/bib_ref] [bib_ref] Latent period for malignant mesothelioma of occupational origin, Lanphear [/bib_ref]. MPM is most commonly diagnosed once symptoms manifest, or at least with major radiographic abnormalities. According to 2018 SEER data, almost three times as many patients presented with distant spread as compared with localized disease. Patients have an average survival rate of seven to twelve months from the time of diagnosis of MPMs [bib_ref] Clinical diagnosis of malignant pleural mesothelioma, Bianco [/bib_ref]. We report here a very unusual case where a patient's localized, completely resectable desmoid sarcomesothelioma was detected on a screening low-dose CT (LDCT) scan. ## Case presentation The patient is a 71-year-old female, a current 56-pack-year cigarette smoker with a past medical history of myocardial infarction and stroke, with a Zubrod score of zero. She denied fevers, chills, night sweats, hemoptysis, or unexpected weight loss and did not have occupational exposure to asbestos. A screening LDCT revealed a 1.9 cm × 1.8 cm × 1.4 cm right lower lobe lesion with smooth margins and close association with the hemidiaphragm . A whole-body positron emission tomography (PET) scan showed mild hypermetabolic activity within the lesion with a maximum standard uptake value (SUV) of 2.6 without evidence of nodal involvement. CT-guided biopsy was inconclusive, with findings of a small amount of fibrous scar-like tissue with chronic inflammation mixed with mesothelial lined tissue, alveolar lung parenchyma, and striated muscle. ## Figure 3: pet scan Hypermetabolic activity within the lesion with a maximum SUV of 2.6 without evidence of nodal involvement Given the overall clinical history and imaging, it was decided that the patient required additional biopsy via either a repeat CT-guided needle or surgical excision. Because of the proximity to the diaphragm, a repeat needle biopsy was considered too difficult, and she was referred to a cardiothoracic surgeon. Right, video-assisted thoracoscopic surgical (VATS) wedge resection of the mass, incorporating small but adequate margins of lung and diaphragmatic muscle, was performed [fig_ref] FIGURE 5: Image of mass after staple placementImage of the mass during video-assisted thoracoscopic... [/fig_ref]. Because lymph nodes were neither enlarged nor "hot" on the PET scan, they were not sampled. The intraoperative frozen section was able to confirm the presence of malignancy without further identification, which would require additional studies. The margins were negative for a tumor on a frozen section. ## Figure 4: mass prior to resection Image of the mass during video-assisted thoracoscopic surgery showing the mass prior to resection The patient was referred to a medical oncologist; the patient opted for imaging surveillance, and at 12 months has shown no evidence of tumor recurrence on PET/CT. # Discussion The above case is notable because it shows that LDCT scans discover cancers and save lives. This is also demonstrated by National Lung Screening Trial data showing a 20% relative reduction in mortality from non-small cell lung cancer with LDCT compared to chest X-rays [bib_ref] Reduced lung-cancer mortality with low-dose computed tomographic screening, Aberle [/bib_ref]. Second, this case highlights the novelty of small and resectable sarcomatoid mesotheliomas, which have been described only in individual case reports. Relatedly, the case presented a dilemma for the patient and her oncologist because standard guidelines, including the National Comprehensive Cancer Network (NCCN), do not define a recommended treatment due to the rarity of resectable sarcomatoid types. While she was offered adjuvant ipilimumab and nivolumab, as approved by the United States Food and Drug Administration (FDA) as adjuvant therapy, she decided for ongoing screening [bib_ref] First-line nivolumab plus ipilimumab in unresectable malignant pleural mesothelioma (CheckMate 743): a..., Baas [/bib_ref]. A review of the literature also indicates that approaches such as chemotherapy with tumor treating fields (TTF) or pegylated arginine deaminase (ADI-PEG 20) are possible therapeutic approaches for mesothelioma [bib_ref] Tumour Treating Fields in combination with pemetrexed and cisplatin or carboplatin as..., Ceresoli [/bib_ref]. Although it is difficult to estimate the patient's expected prognosis, it is documented that the five-year survival rate of localized malignant pleural mesothelioma (MPM) is 20%, in contrast to 8% for distant MPM. However, these data likely reflect cases involving larger tumors in which the patient undergoes larger, more invasive surgeries. The final unusual piece of data, in this case, is the lack of any evidence of prior exposure to asbestos. # Conclusions This case highlights the importance of LDCT scanning for early diagnosis of life-threatening illnesses, based on USPSTF guidelines. Also emphasized is the clinical decision-making that prompted the patient's caretakers to persist in securing a diagnosis based on her risk factors. Given the rare nature of sarcomatoid mesothelioma, we hope the information provided can add to the few other case reports for a better understanding of this cancer. # Additional information disclosures Human subjects: Consent was obtained or waived by all participants in this study. Conflicts of interest: In compliance with the ICMJE uniform disclosure form, all authors declare the following: Payment/services info: All authors have declared that no financial support was received from any organization for the submitted work. Financial relationships: All authors have declared that they have no financial relationships at present or within the previous three years with any organizations that might have an interest in the submitted work. Other relationships: All authors have declared that there are no other relationships or activities that could appear to have influenced the submitted work. [fig] FIGURE 5: Image of mass after staple placementImage of the mass during video-assisted thoracoscopic surgery showing the mass with the staple in place Final pathologic analysis revealed a 2 cm high-grade sarcomatoid mesothelioma with extensive desmoplastic morphology and negative margins. The immunostains were positive for calretinin, MCK, GATA3, patchy positivity for CK7 and pan keratin, and rare cells positive for WT-1. [/fig]

SLAMF8 Participates in Acute Renal Transplant Rejection via TLR4 Pathway on Pro-Inflammatory Macrophages # Introduction Kidney transplantation is the most optimal renal replacement therapy both for the quality and quantity of life that it provides and for cost effectiveness compared to classic maintenance dialysis for patients in end-stage renal disease (ESRD) [bib_ref] World Kidney Day Steering Committee 2012. The Global Role of Kidney Transplantation, Garcia [/bib_ref]. The overall risk of acute rejection within 1 year after transplantation has been steadily decreasing to <15% with the introduction of several newer immunosuppressive agents. Nevertheless, short-term improvement in graft survival decreased since 2000, and disappointingly, long-term improvement remained unchanged [bib_ref] Rejection of the Kidney Allograft, Nankivell [/bib_ref] [bib_ref] Analyses of the Short-and Long-Term Graft Survival After Kidney Transplantation in Europe..., Coemans [/bib_ref] [bib_ref] Long-Term Survival After Kidney Transplantation, Hariharan [/bib_ref]. Timing of acute rejection (AR) and the number of episodes still remains a major risk factor for the development of chronic renal allograft failure (CAF), which is a major cause of late graft loss [bib_ref] Acute Rejection and Chronic Nephropathy: A Systematic Review of the Literature, Wu [/bib_ref] [bib_ref] Collaborative Transplant Study Report, Opelz [/bib_ref]. AR consists of two distinct diseases: T-cell-mediated rejection (TCMR) characterized by arteritis, interstitial inflammation, and tubulitis, which is the main type in the first year of AR and antibody-mediated rejection (ABMR) refined to encompass histological evidence of capillaritis and serological evidence [bib_ref] The Banff 2019 Kidney Meeting Report (I): Updates on and Clarification of..., Loupy [/bib_ref] [bib_ref] The Banff 2017 Kidney Meeting Report: Revised Diagnostic Criteria for Chronic Active..., Haas [/bib_ref]. However, as far as we know, the pathophysiology of AR is multifactorial and still not fully defined. Therefore, there is a continuing need to screen new biomarkers for the diagnosis and treatment of allograft rejection after kidney transplantation. Transcriptional genomic information to acute allograft rejection after renal transplantation has shed new light on our understanding of the pathogenesis [bib_ref] The Use of Genomics and Pathway Analysis in Our Understanding and Prediction..., Menon [/bib_ref]. Weighted gene coexpression network analysis (WGCNA) has been applied to many important studies such as cancer [bib_ref] Molecular Signature of Subtypes of Non-Small-Cell Lung Cancer by Large-Scale Transcriptional Profiling:..., Niemira [/bib_ref] [bib_ref] Pan-Cancer Analysis Identifies Telomerase-Associated Signatures and Cancer Subtypes, Luo [/bib_ref] , autoimmune diseases [bib_ref] Identification and Validation of IFI44 as Key Biomarker in Lupus Nephritis, Shen [/bib_ref] [bib_ref] Identifying Key Genes and Functionally Enriched Pathways in Sjögren's Syndrome by Weighted..., Yao [/bib_ref] , and neurodegenerative diseases [bib_ref] Application of Weighted Gene Co-Expression Network Analysis to Explore the Key Genes..., Liang [/bib_ref] [bib_ref] Metabolic Alterations in Multiple Sclerosis and the Impact of Vitamin D Supplementation, Bhargava [/bib_ref] since its introduction in 2005. WGCNA can potentially identify the gene network significantly involved in AR, and hub gene in estimating network structures can improve the performance of the predicting biological processes and gene regulation to get deep understanding of its pathogenesis [bib_ref] Enhanced Construction of Gene Regulatory Networks Using Hub Gene Information, Yu [/bib_ref]. Two recent studies identified several genes associated with kidney transplant rejection via WGCNA based on peripheral blood lymphocytes (PBLs) or peripheral blood (PB) [bib_ref] Might be Important Biomarkers in Kidney Transplant Rejection, Fan [/bib_ref] [bib_ref] Identification of Biomarkers for Predicting Allograft Rejection Following Kidney Transplantation Based on..., Wang [/bib_ref]. Nevertheless, there are few relative studies on kidney transplantation based on percutaneous allograft biopsy. Due to the latest advances in basic science, macrophages serve as crucial mediators of acute and chronic allograft immunopathology. It is well known that macrophages can trigger an adaptive immune response, persist T-cell-mediated rejection and antibody-mediated rejection, and promote allograft fibrosis [bib_ref] The Evolving Roles of Macrophages in Organ Transplantation, Li [/bib_ref]. Renal macrophages exhibit a pro-inflammatory phenotype signature for interferon gamma (IFNg) activated and secrete a variety of cytokines, which can activate endothelial cells and promote the production of cytotoxic T cells during acute TCMR associated with poor allograft outcomes [bib_ref] Landscape of Innate Immune System Transcriptome and Acute T Cell-Mediated Rejection of..., Mueller [/bib_ref]. Therefore, macrophages have important effects on transplantation results. However, the exact mechanisms controlling macrophage functions are not yet completely understood. In this study, by using WGCNA-based methods, we downloaded the Gene Expression Omnibus database GSE138043 and screened six hub genes related to the AR. Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis was performed to reveal pathways in target module, which possibly influence the pathogenesis of AR, and Gene Set Enrichment Analysis (GSEA) was performed to show enrichment results of differentially expressed genes in six hub genes high-expression groups. In addition, we utilized PBMC from patient in whom acute rejection occurred after surgery in our hospital to validate six hub genes. We performed single-cell RNA sequencing to further study the cell types changes related to AR. The AR patient had a higher percentage of native T, CD1C+_B DC, NKT, NK, and monocytes. Immunohistochemistry of SLAMF8 revealed that SLAMF8 + cells infiltrated in the human allograft tissue in AR. We constructed Immunofluorescence staining of SLAMF8 and TLR4 to validate that SLAMF8 was involved in the proinflammatory macrophages via TLR4, which contributed to AR process in vivo and in vitro. # Materials and methods ## Data collection and preprocessing We downloaded mRNA expression profiles of human AR from the Gene Expression Omnibus (GEO) database. In our study, GSE138043 was used to construct co-expression networks and identify hub genes related to AR. The microarray dataset provided gene expression profile in the percutaneous allograft biopsy from 15 AR patients and 37 NAR [bib_ref] Key Driver Genes as Potential Therapeutic Targets in Renal Allograft Rejection, Yi [/bib_ref]. According to the data processing information of GSE138043, each dataset was normalized independently using Robust Multiarray Average (RMA) followed by log2 transformation and quantile normalization. Data from GSE50058 and GSE343 were used for hub genes validation. In the GSE50058 dataset, 42 AR patients and 58 STA individuals were recruited, and the RNA was extracted from their renal allograft biopsy. In the GSE343 dataset, the total RNA was extracted from the kidney tissue of 25AR patients and 15 NAR. Supplementary shows the summary of discovery and validation microarray data sets of clinical biopsy samples from kidney transplants. ## Differentially expressed genes screening "limma" R package was utilized to the differentially expressed screen genes (DEGs) between AR and NAR in the expressing data. The genes with adjusted p-value <0.05 were selected as having significant change. "ggplot2" and "pheatmap" were used respectively to paint the volcano plot and heatmap of all DEGs. ## Construction of co-expression network The co-expression network of the genes was constructed based on GSE138043 microarray dataset by the R package "WGCNA." The soft-thresholding power that we chose was 17 when 0.9 was used as the correlation coefficient threshold. We defined 0.25 as the threshold for cut height to merge possible similar modules. # Functional enrichment analysis To obtain further insights into the function of the target module most related to AR, we referred to the Database for Annotation, Visualization and Integrated Discovery (DAVID) (https://david. ncifcrf.gov/) to perform the KEGG enrichment analysis. The results were shown graphically by the R package "ggplot2." ## Hub genes identification The green module, which was most significantly related to AR, was imported into Cytoscape with their weighted correlations. We identified the hub gene with the following criteria: (1) DEGs in green module; (2) gene significance (GS) > 0. [bib_ref] Rejection of the Kidney Allograft, Nankivell [/bib_ref] ## Single-cell rna sequencing A 10X Genomics Chromium machine was used for single-cell capture and cDNA preparation following manufacture's instruction. Chromium ™ Single Cell 3′ Solution was used to perform reverse transcription on gel bead in emulsion, followed by cDNA cleanup and amplification. The cDNA is digested and broken into fragments of about 200-300 bp, followed by the traditional second-generation sequencing library construction process, and PCR amplification is performed to obtain a DNA library. Illumina sequencing platform of paired-end sequencing mode was used to perform high-throughput sequencing on the established library. Sequence data were processed with Cell Ranger V2.1.0 (10X Genomics). ## Quality control Then, quality control was performed to filter low-quality cells. For 10X-derived datasets, we only retained cells that had (1) genes more than 200 and <6,000, (2) UMIs more than 500 and <40,000, and (3) <15% of reads mapped to mitochondrial genes. ## Clinical validation The Nephroseq v5 online database (http://v5.nephroseq.org/), an integrated data-mining platform for gene expression data sets of kidney diseases, was adopted to validate the correlation between the hub genes and clinical manifestations of AR by Spearman rank correlation coefficient analysis. A p-value of <0.05 was considered statistically significant. r>0.6 was considered strong correlation, and 0.4<r<0.6 was considered medium intensity correlation. # Cell types analysis The xCell (https://xcell.ucsf.edu/), which is a webtool that performs cell-type enrichment analysis from gene expression data for 64 immune and stroma cell types, was adopted to reveal different infiltrating cell types between AR and NAR, and adjusted p-value < 0.01 was chosen as the cutoff criterion. Celltype enrichment score is shown in . To further explore the six hub genes expression in 76 single cell types, we obtained RNA expression values per cell types from Human Protein Atlas Dataset (proteinatlas.org). Single cell-type clusters were normalized separately from other transcriptomics datasets using trimmed mean of M values (TMM). To generate expression values per cell type, clusters were aggregated per cell type by first calculating the mean nTPM in all cells with the same cluster annotation within a dataset. ## Gene set enrichment analysis To further explore the potential function of the selected hub genes in AR, we used gene set enrichment analysis (GSEA_4.1.0) for single hub gene. In the dataset GSE138043, samples were divided into two groups according to the median expression level of hub genes. The h.all.v6.2.sytmbols.gmt in Molecular Signatures Database (MSigDB) was selected as the reference gene set, and adjusted p-value <0.05 was considered significantly different. The results were showed graphically by the R package "ggplot2." ## Cell culture The Raw264.7 cells were cultured in Dulbecco's modified Eagle's medium (DMEM) High Glucose (11965084, Gibco) containing 10% fetal bovine serum (FBS) (12103C, Sigma) and 1% penicillin-streptomycin (15070063,Gibco), incubated at 37°C in a humidified atmosphere containing 5% CO 2 , and routinely passaged every 1 or 2 days. To induce RAW264.7 cell line to M1/ M2 phenotypes, 10 5 Raw264.7 cell were seeded in six-well plates 24 h before exposed to IFN-g (20 ng/ml) + lipopolysaccharide (LPS) (10 ng/ml) to M1 phenotype, and IL-4 (10 ng/ml) to M2 phenotype for 24 h. ## Bone-marrow-derived macrophages isolation and culture Isolation and culture of bone-marrow-derived macrophages (BMDMs) were described by Pineda-Torra et al. [bib_ref] Isolation, Culture, and Polarization of Murine Bone Marrow-Derived and Peritoneal Macrophages, Pineda-Torra [/bib_ref]. In brief, bone marrow was flushed out from the femurs and tibias, cultured and differentiated for 7 days in 1640 supplemented with 10% heat-inactivated FBS and 1% penicillin-streptomycin with macrophage-stimulating factor (M-CSF) (50 ng/ml) at 37°C in a humidified incubator with 5% CO 2 . ## Flow cytometry to measure macrophage polarization Flow cytometry was used to measure the phenotypical changes in Raw264.7 macrophages. Single-cell suspension is prepared before staining with fluorochrome-labeled anti-CDF4/80 (clone BM8, BioLegend, San Diego, CA, USA), anti-CD80 (clone 16-10A1, eBioscience, San Diego, CA, USA). Data were analyzed using FlowJo v.10 (Treestar, Ashland, OR). ## Blood sample and the percutaneous allograft biopsy collection Research involving human participants was reviewed and approved by the Research Ethics Committee of the First Affiliated Hospital of Zhejiang University School of Medicine. Patients/participants (or their close relatives) provided written informed consent to participate in this study. Peripheral blood mononuclear cell (PBMC) was isolated within 3 h after collection. Percutaneous allograft biopsy was collected and fixed with 4% formalin for paraffin embedding. Biopsies were scored by the revised Banff 2019 classification of renal allograft pathology; rejection cases here were TCMR including borderline cases. Supplementary showed the characteristics of the samples used in this study. ## Rna extraction and real-time quantitative pcr Total RNA was extracted from cultured cells and peripheral blood mononuclear cells (PBMCs) by Trizol reagent (Invitrogen, CA, USA). cDNA was prepared using the PrimeScriptTM RT Reagent Kit with gDNA Eraser (No. RR047A, Takara, Shiga, Japan) following the manufacturer's protocol. Real-time PCR was run using SYBR Green and CFX96 ™ Real-Time PCR Detection Systems (Bio-Rad, CA, USA). The mRNA levels of selected genes were calculated after normalization to b-actin by using the 2 −DDCt method according to the manufacturer's protocol. All primer sequences used are shown in Immunohistochemistry Immunohistochemistry (IHC) was performed following standard protocol. Briefly, after being dewaxed and rehydrated, the 2-mm paraffin-embedded sections were incubated with Anti-BLAME Polyclonal Antibody (bs-2473R, Bioss, Boston, MA, USA). After washing, the sections were incubated with the horseradish-peroxidase-labeled anti-mouse/rabbit IgG polymer (GK500710, GeneTech, South San Francisco, CA, USA) and diaminobenzidine. The sections were then counterstained with hematoxylin, dehydrated, and cleared. Six random fields of each section were photographed, and the staining was semi-quantified using the National Institutes of Health Image J by an investigator blinded to the experimental protocol. Immunofluorescence Assessment of Cultured Raw264.7 Coverslips containing RAW264.7 or BMDMs were fixed with 4% paraformaldehyde for 15 min and blocked in phosphate-buffered saline (PBS) containing 0.1% Triton X-100 and 3% bovine serum albumin(BSA) for 30 min at room temperature prior to incubation with Anti-BLAME Polyclonal Antibody (bs-2473R, Bioss), TLR4 antibody (sc-293072, Santa Cruz) overnight in a humidified chamber at 4°C. Slips were incubated with secondary antibody Alexa Fluor 594-conjugated anti-rabbit IgG (1:500) and Alexa Fluor 488-conjugated anti-mouse IgG (1:500) 1 h at 37°C. Sections were then examined by immunofluorescence microscopy (Leica DMLB, Wetzlar, Germany). ## Western blotting Total RAW264.7 cells were lysed in radioimmunoprecipitation assay (RIPA) cell lysis buffer following separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to 0.22-mm polyvinylidene fluoride membranes (Millipore, Burlington, MA, USA). Blocked with 5% milk and incubated with the inducible nitric oxide synthase (iNOS) antibody (ab178945, Abcam, Cambridge, UK) at 4°C overnight. Protein was visualized using secondary anti-rabbit IgG or anti-mouse IgG (Sigma) with conjugated horseradish peroxidase and chemiluminescent substrate (Millipore, Billerica, MA, USA). # Statistical analysis The statistical significance differences between the two groups were analyzed using non-parametric test or t-test based on data distribution characteristics. All analyses were conducted using software R4.1.0. A p-value < 0.05 was considered statistically significant. # Results ## Differentially expressed genes between ar and non-ar controls Kidney transcriptome data from GSE138043 containing 15 AR patients (rejection at 12 months post-renal transplant) and 37 NAR (non-rejection at 12 months post-renal transplant) was used for further analysis. We identified 1,561 DEGs (differential genes, adjusted p-values < 0.05) between the AR and NAR groups. Among 1,561 DEGs, 541 genes were upregulated, while 1,020 genes were downregulated. All genes are displayed in volcano plot in , and DEGs are listed in . Additionally, the red plots represent adjusted p < 0.01, orange plots represent log2FC > 1, and green plots that were annotated represent both. Unsupervised clustering hierarchy was used in heatmap . ## Weighted co-expression network construction WGCNA package was applied to compile the network. Keeping to the scale-free topology criterion, b = 17 was considered in this study for which the fit index curve flattens out upon reaching a high value (>0.9) and the mean connectivity ≤100 . Hierarchical clustering was used to generate a hierarchical clustering dendrogram of genes; meanwhile, Dynamic Tree Cut R library was used for detecting clusters. As shown in Figures 2B-D, 12 distinct gene modules (M1-12) were defined, as MEDissThres was set to 0.25 to merge similar modules. Genes failing to fit within a distinct group were assigned to the gray module. The interaction relationships of 1,000 randomly selected genes were presented in the network heatmap (Supplementary .The genes in the same module were highly correlated, while they were weakly correlated to those in other modules. Thus, the reliability of the modules was verified. ## Identification of meta-modules and hub genes associated with ar Module-trait correlations analyses showed that multiple modules were related to AR The summary of significance of all genes in each module related to AR is shown in . Clearly, the green module was most significantly related to AR, followed by the turquoise module. shows the significance of these genes in the green module for AR (cor = 0.35, p = 2.4e−22). To investigate the potential biological function of genes in the green modules, we performed KEGG enrichment analysis. The top 20 pathways terms of green module are shown in . We found that genes in green modules played roles in cytokine-cytokine receptor interaction, chemokine signaling pathway, cell adhesion molecules (CAMs), tuberculosis, and other important regulatory processes. To identify hub genes in the target module, we calculated the MCC values via Cytohubba and constructed a network based on the top 10 genes (Supplementary . The module was visualized using Cytoscape 3.8 software . The node colors coded from yellow to red (low to high) correspond to the top 10 MCC values from low to high. In intersection with GS > 0.4, MM > 0.9, and DEGs in the green module, six genes were regarded as the hub genes , including dedicator of cytokinesis 2 (DOCK2), NCK-associated protein 1 like (NCKAP1L), interleukin 2 receptor subunit gamma (IL2RG), SLAM family member 8 (SLAMF8), CD180 molecule (CD180), and protein tyrosine phosphatase receptor type E (PTPRE). Relative mRNA expression of six hub genes in GSE138043 is shown in . ## Geo, clinical, and qrt-pcr validation As expected, the expression levels of hub genes including DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE were significantly upregulated in AR samples from the GSE50058 dataset [fig_ref] FIGURE 4 |: GEO and clinical validation [/fig_ref]. For verifying hub genes, we obtained another dataset, GSE343, and analyzed the expression levels of the above five genes except for SLAMF8, which was not found in this dataset between AR patients and STA patients [fig_ref] FIGURE 4 |: GEO and clinical validation [/fig_ref]. The expression of these genes was also upregulated in AR samples. We used the Nephroseq v5 online database to explore the correlation between the expression of IL2RG and clinical traits of AR. As shown, there was a positive correlation between the expression of IL2RG in AR with the Banff pathological grading of transplanted kidney (r=0.6540, p<0.0001) [fig_ref] FIGURE 4 |: GEO and clinical validation [/fig_ref].Correlation between the expression of DOCK2 in AR and the Banff pathological grading is shown in [fig_ref] FIGURE 4 |: GEO and clinical validation [/fig_ref]. To study the transcriptional changes related to AR further, we . In addition, to further validate these six hub genes, we collected PBMCs from eight non-AR and 10 AR patients to perform qRT-PCR. The results showed that compared with NAR group, the mRNA levels of DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE were all significantly elevated in AR patients [fig_ref] FIGURE 5: | scRNA-seq in patient PBMCs of acute rejection post renal transplant and... [/fig_ref]. # Cell-type enrichment analysis Cell-type enrichment analysis was performed by xCell from gene expression data for 64 immune and stroma cell types. Our data revealed that the AR group had higher immune scores and microenvironment scores than the NAR group. Among the 64 cell types, scores of 20 types of cells were significantly differently expressed (p<0.01), including immune cells, such as B cells, basophils, CD4+ Tem, CD4+ memory T cells, CD8+ T cells, CD8+ Tcm, class-switched memory B cells, DC, macrophages, M1, mast cells, memory B cells, monocytes, plasma cells, aDC, cDC, naive B cells, pDC, and stroma cells such as endothelial cells, smooth muscle, and MV endothelial cells . We used the Protein Atlas online database to explore the six hub genes RNA expression in 76 single-cell types (Supplementary . Except for SLAMF8, which is more singly expressed in Langerhans cell and macrophages, the other five genes are more widely expressed in immune cells such as B cells, DC, T cells, and monocyte consistent with the different cell types in AR groups, meaning that six hub genes exercise certain biological properties in these cell types during AR. PTPRE, including "allograft rejection", "interferon g response", "interferon a response", and "inflammatory response". Moreover, gene sets "IL6-JAK-STAT3 signaling", "KRAS signaling", and "TNFa signaling via NFkB" were enriched in the samples with either DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE highly expressed. ## Gene set enrichment analysis ## Slamf8 participate in ar progression via tlr4 in vivo SLAMF8, one of the six hub genes, was also the top rejectionassociated transcripts in TCMR versus everything else including ABMR in previous study [bib_ref] Comprehensive Analysis of Transcript Changes Associated With Allograft Rejection: Combining Universal and..., Halloran [/bib_ref]. In addition, SLAMF8 is more specifically expressed in macrophages, which is exactly the cell type that differentially infiltrated in the AR group in our above research results ; Supplementary . Immunohistochemical staining of kidney tissue revealed a higher level of SLAMF8 expression in the renal interstitium from TCMR (T-cell-mediated acute rejection after renal transplantation) kidney tissue than from HC (healthy donor control) [fig_ref] FIGURE 8 |: SLAMF8 participate in AR progression via TLR4 in vivo [/fig_ref]. A previous study confirmed that SLAMF8 maintained TLR4 expression on macrophages and promoted LPS-induced mitogen-activated protein kinase (MAPK) activation [bib_ref] Combined Deficiency of SLAMF8 and SLAMF9 Prevents Endotoxin-Induced Liver Inflammation by Downregulating..., Zeng [/bib_ref]. We subsequently determined whether the SLAMF8 participates in macrophage activation via TLR4. Fluorescence detection of SLAMF8 and TLR4 revealed that approximately complete SLAMF8+ cells express TLR4 [fig_ref] FIGURE 8 |: SLAMF8 participate in AR progression via TLR4 in vivo [/fig_ref]. Thus, we speculated that SLAM8 participates in AR progression, likely through upregulating TLR4 expression. ## Slamf8 and tlr4 are co-expressed in m1-type macrophages In order to explore in which phenotype of macrophages SLAMF8 is specifically expressed, murine RAW 264.7 macrophages and primary BMDM cells were treated with 10 ng/ml LPS plus 20 ng/ ml IFNg or 10 ng/ml IL-4 for 24 h. CD80 and CD206 are known to be a specific surface marker of the M1 phenotype and the M2 phenotype, respectively. Flow cytometry and RT-qPCR were used to measure the polarization of RAW 264.7 macrophages [fig_ref] FIGURE 4 |: GEO and clinical validation [/fig_ref]. Western blotting using the M1 phenotype marker iNOS was confirmed to be increased in the LPS plus IFNg treated group in RAW264.7 (Supplementary [fig_ref] FIGURE 4 |: GEO and clinical validation [/fig_ref]. The mRNA expression of SLAMF8 and TLR4 was increased in LPS and IFNg treated RAW 264.7 macrophages and BMDMs [fig_ref] FIGURE 5: | scRNA-seq in patient PBMCs of acute rejection post renal transplant and... [/fig_ref] consistent with the phenomenon in immunofluorescence staining [fig_ref] FIGURE 5: | scRNA-seq in patient PBMCs of acute rejection post renal transplant and... [/fig_ref]. Two-color immunofluorescence analysis revealed that the M1 phenotype contained large numbers of TLR4-expressing SLAMF8+ cells in contrast to M0-and M2-type macrophages [fig_ref] FIGURE 5: | scRNA-seq in patient PBMCs of acute rejection post renal transplant and... [/fig_ref]. # Discussion Even if the incidence of clinical acute rejection and subclinical rejection in the first year after kidney transplantation is controlled in 10%-15%, respectively, preventing acute rejection remains the key to achieving long-term graft survival [bib_ref] Long-Term Survival After Kidney Transplantation, Hariharan [/bib_ref]. Therefore, timely detection of rejection is important for the surveillance after transplantation. To our knowledge, there are few studies analyzing kidney transplant rejection using WGCNA [bib_ref] Might be Important Biomarkers in Kidney Transplant Rejection, Fan [/bib_ref] [bib_ref] Identification of Biomarkers for Predicting Allograft Rejection Following Kidney Transplantation Based on..., Wang [/bib_ref] [bib_ref] Correlating Transcriptional Networks to Acute Rejection in Human Kidney Transplant Biopsies, Liu [/bib_ref]. This study shows that co-expression network analysis was employed to mine the hub gene based on expression in the percutaneous allograft biopsy of 15 AR and 37 NAR. Through WGCNA, we divided all genes into 12 separate modules and found that the green module was the most related to AR. Six hub genes were then screened out satisfying three criteria: (1) DEGs; (2) GS > 0.4, MM > 0.9; and (3) the 10 highest MCC value, followed by validation in both two datasets and clinical traits of AR. Of the six hub genes derived, some have well-described functions in the immune response (e.g., DOCK2, IL2RG, PTPRE, and CD180), while others have not been as well characterized (e.g., NCKAP1L and SLAMF8) and represent opportunities for future study. DOCK2, a member of the CDM protein family, plays a critical role in lymphocyte homing and immunological synapse formation by remodeling the actin cytoskeleton in response to chemokine signaling [bib_ref] Haematopoietic Cell-Specific CDM Family Protein DOCK2 Is Essential for Lymphocyte Migration, Fukui [/bib_ref]. DOCK2 mediates GTP-GDP exchange reaction for Rac through its DOCK homology region (DHR)-2 (also known as Docker) domain [bib_ref] Multiple Factors Confer Specific Cdc42 and Rac Protein Activation by Dedicator of..., Kulkarni [/bib_ref]. Mutations in this gene result in immunodeficiency, a combined form of immunodeficiency that affects T-cell number and function, also with variable defects in B-cell and NK-cell function [bib_ref] Human DOCK2 Deficiency: Report of a Novel Mutation and Evidence for Neutrophil..., Moens [/bib_ref] [bib_ref] Inherited DOCK2 Deficiency in Patients With Early-Onset Invasive Infections, Dobbs [/bib_ref]. Deletion of DOCK2 suppresses cardiac allograft rejection [bib_ref] Deletion of DOCK2, a Regulator of the Actin Cytoskeleton in Lymphocytes, Suppresses..., Jiang [/bib_ref]. IL2RG, interleukin 2 receptor subunit gamma chain, or CD132, is the co-receptor subunit of a variety of important immune factors, including IL-2, IL-4, IL-7, IL-9, IL-15, and IL-21. Therefore, it is also called the receptor common gamma chain (gc) [bib_ref] The Common Cytokine Receptor g Chain Family of Cytokines, Lin [/bib_ref]. In mammals, the IL2Rg gene is located on the X chromosome. Its mutation called X-linked severe combined immunodeficiency (X-SCID), presenting with absent or profoundly diminished peripheral T and NK cells and functionally defective B cells [bib_ref] Humanized Mouse Models of Clinical Disease, Walsh [/bib_ref] [bib_ref] New Insights Into the Regulation of T Cells by Gamma(C) Family Cytokines, Rochman [/bib_ref]. In severe combined immunodeficiency (SCID) gene homozygous mutation or recombination activation gene 1 (Rag1) or Rag2 homozygous mutation mice, accompanied by mutation of the interleukin 2 receptor gamma chain (IL2Rg) locus, comparing with previous immunodeficiency mouse models, the implantation and function of human hematopoietic stem cells (HSC) and peripheral blood mononuclear cells (PBMC) are greatly increased [bib_ref] Humanized Mice in Translational Biomedical Research, Shultz [/bib_ref]. In this study, we found that DOCK2 and IL2RG mRNA levels correlated to inflammatory parameters according to the Banff classification. The nine receptors of the SLAM family are differentially expressed on the surface of hematopoietic cells such as thymocytes, memory CD4+ and CD8+T cells, dendritic cells, monocytes, macrophages, and platelets. They regulate not only the proliferation, cytotoxicity, and cytokine production of T lymphocytes but also the lytic activity, cytokine production, and MHC-independent inhibition of natural killer (NK) cells; B cell activation and proliferation; regulation of neutrophil; and macrophage killing and platelet aggregation [bib_ref] Immune Regulation by SLAM Family Receptors and SAP-Related Adaptors, Veillette [/bib_ref]. In contrast to the classical SLAMF receptors, SLAMF8 have no signaling motifs including the ITSM in their short cytoplasmic tail. Limited studies have indicated that combined deficiency of SLAMF8 and SLAMF9 prevents endotoxin-induced liver inflammation by downregulating TLR4 expression on macrophages [bib_ref] Combined Deficiency of SLAMF8 and SLAMF9 Prevents Endotoxin-Induced Liver Inflammation by Downregulating..., Zeng [/bib_ref] , and SLAMF8 can negatively regulate ROS production by macrophages [bib_ref] Cutting Edge: Slamf8 Is a Negative Regulator of Nox2 Activity in Macrophages, Wang [/bib_ref]. In addition, a previous study has shown that SLAMF8 is the top differentiating transcripts rejectionassociated transcripts in TCMR versus everything else including ABMR, and SLAMF8 is the most specific transcript in macrophages cell lines for TCMR [bib_ref] Comprehensive Analysis of Transcript Changes Associated With Allograft Rejection: Combining Universal and..., Halloran [/bib_ref] [bib_ref] Molecular Landscape of T Cell-Mediated Rejection in Human Kidney Transplants: Prominence of..., Venner [/bib_ref]. Bone marrow cells follow a differentiation trajectory from monocytes to proinflammatory macrophages and become predominantly infiltrating cells in the intimal arteritis of biopsies graded as Banff II or III acute allogeneic renal rejection [bib_ref] The Macrophage Is the Predominant Inflammatory Cell in Renal Allograft Intimal Arteritis, Matheson [/bib_ref] [bib_ref] Single Cell Transcriptomics of Mouse Kidney Transplants Reveals a Myeloid Cell Pathway..., Dangi [/bib_ref]. Due to SLAMF8 mainly expressed on macrophages and the important role of macrophages in transplant rejection, we further investigated the expression of SLAMF8 in from TCMR in vivo and macrophages of different phenotypes in vitro. We observed that SLAMF8 expressed highly in TCMR than HC and in M1 phenotype. TLR4 is a member of the toll-like receptor family and plays an important role in regulating innate immunity in response to exogenous and endogenous molecular patterns [bib_ref] Toll-Like Receptor Signaling Pathways, Barton [/bib_ref]. Activation of the innate immunity through toll-like receptors (TLRs) has been postulated to play an important role in the pathophysiology of renal allograft dysfunction [bib_ref] Intragraft Toll-Like Receptor Profiling in Acute Renal Allograft Rejection, Dessing [/bib_ref]. Renal transplant recipients with TLR4 polymorphism present a lower risk of acute allograft rejection and lower rates of delayed graft function as compared to those with normal TLR4 function [bib_ref] Relevance of Toll-Like Receptor-4 Polymorphisms in Renal Transplantation, Ducloux [/bib_ref] , also consistent with the observation that acute kidney allograft rejection was modestly attenuated in TLR4 −/− mice [bib_ref] Toll-Like Receptor 4 Deficiency Improves Short-Term Renal Function But Not Long-Term Graft..., Kwan [/bib_ref]. Previous reports have demonstrated that activation of TLR4 triggers a phenotypic switch of macrophages from a quiescent population to an inflammatory population and inhibition of TLR4 suppressed macrophages polarization [bib_ref] Curcumin Modulates Macrophage Polarization Through the Inhibition of the Toll-Like Receptor 4..., Zhou [/bib_ref] [bib_ref] Toll-Like Receptor 4 Plays a Key Role in Advanced Glycation End Products-Induced..., Liu [/bib_ref] [bib_ref] Ginseng-Derived Nanoparticles Alter Macrophage Polarization to Inhibit Melanoma Growth, Cao [/bib_ref]. Because SLAMF8 has no signaling activity and the TLR4 has essential role in macrophages and immune rejection, we wondered whether SLAMF8 participates in macrophage activation via TLR4. Here, we identified that approximatively complete SLAMF8+ cells express TLR4 in TCMR. M1 macrophage contained large numbers of TLR4-expressing SLAMF8+ cells in contrast to M0and M2-type macrophages. In summary, our study finds involvement of the key gene co-expression module, hub genes, and some functional biological pathways related to "interferon g response", "interferon a response", and "inflammatory response" in the pathogenesis of AR. SLAMF8 was highly expressed in proi n fl a m m a t o r y m a c r o p h a g e -m e d i a t e d a c u t e r e n a l transplantation rejection accompanied by TLR4 high expression, and it presents a potentially novel therapeutic target for controlling kidney allograft rejection and improving kidney allograft survival. These findings provide new insights into the development of AR, although the exact molecular mechanism of hub genes and functional pathway in AR still need to be further explored. # Data availability statement The data presented in the study are deposited in the OMIX, China National Center for Bioinformation / Beijing Institute of Genomics, Chinese Academy of Sciences (https://ngdc.cncb.ac. cn/omix/release/OMIX873), accession number OMIX873. # Ethics statement The studies involving human participants were reviewed and approved by the Research Ethics Committee of the First Affiliated Hospital, College of Medicine, Zhejiang University. The patients/participants provided their written informed consent to participate in this study. # Author contributions HJ, JC, and LT designed the study. LT and LS analyzed bioinformatics. CW, SF, and YW collected PBMC and the were measured in BMDMs, BMDMs+LPS (10ng/ml)+IFNg (20ng/ml) and BMDMs+IL-4 (10ng/ml) for 24 h. (C-E) Representative and quantification of Immunofluorescence staining of TLR4(green) and SLAMF8(red) in BMDMs, BMDMs+LPS+IFNg and BMDMs+IL-4. Scale bar = 50mm; Six random fields were taken from each coverslip (mean ± SD, n = 6). Supplementary | DEGs in GSE138043. [fig] FIGURE 2 |FIGURE 3 |: performed GSEA, and h.all.v7.4.sytmbols.gmt in MSigDB was used as the reference gene set. The full list of gene sets enriched in samples with DOCK2 (Figure 7A), NCKAP1L (Figure 7B), IL2RG (Figure 7C), SLAMF8 (Figure 7D), CD180 (Figure 7E), or PTPRE (Figure 7F) highly expressed is shown. Four gene sets were enriched in samples with highly expressed DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and Determination of soft-threshold power in the WGCNA. (A) Left: Analysis of the scale-free topology model fit for various soft-threshold powers (b). Right: Analysis of the mean connectivity for various soft-threshold powers. (B) Clustering of module eigengenes. The red line represents MEDissThres=0.25. (C) Dendrogram of all differentially expressed genes clustered based on the measurement of dissimilarity (1-TOM). The branches correspond to modules of highly interconnected groups of genes. (D) The cluster dendrogram and adjacency heatmap of eigengenes. WGCNA revealed gene co-expression networks and the key genes in the percutaneous allograft biopsy of 15 AR patients. (A) Heatmap of the correlation between the module eigengenes and clinical traits of AR. We selected the green block for subsequent analysis. (B) Module significance values of those co-expression modules associated with SS (module significance value indicated the summary of gene significance of all genes in each module, and different colors of column indicated different modules). (C) The gene significance for AR in the green module (one dot represents one gene). (D) Top 20 pathways from Kyoto Encyclopedia of Genes and Genomes enrichment analysis. The x-axis represents KEGG enrichment scores, and the y-axis represents pathway terms. The colors of circle indicate p-values, and the size of circle indicates the numbers of differential RNAs. The redder and larger circle indicates that the enrichment of the pathway is higher and differential RNAs number is larger in the pathway. (E) Interaction of gene co-expression patterns in the green module. Each node corresponds to a gene. Colors from yellow to red correspond to the top 10 maximal clique centrality (MCC) values from low to high. (F) Identification of the hub gene in the intersection of MCC TOP10, DEGs, and GS > 0.4, and MM > 0.9. [/fig] [fig] FIGURE 4 |: GEO and clinical validation. (A) Expression levels of DOCK2, NCKAP1L, IL2RG, SLAMF8, CD180, and PTPRE were significantly upregulated in the renal allograft biopsy of AR patient in dataset GSE50058. (B)The clustering of five hub genes in heatmap of dataset GSE343. (C) Correlation between the expression of IL2RG in AR with the Banff pathological grading of transplanted kidney. STA, stable patients; AR, patient with acute rejection. *p < 0.05, **p < 0.01, ***p < 0.0001. [/fig] [fig] FIGURE 5: | scRNA-seq in patient PBMCs of acute rejection post renal transplant and qRT-PCR validation. (A) Uniform manifold approximation and projection (UMAP) of 26,192 cells, split between P1 and C1. (B) UMAP plot of 19 cell clusters identified based on the expression of highly variable genes. (C) Single-cell RNA sequencing revealed a higher percentage of native T, CD1C+_B DC, NKT, NK, and monocytes in P1 compared with C1. Relative mRNA expression of DOCK2 (D), NCKAP1L (E), IL2RG (F), SLAMF8 (G), CD180 (H), and PTPRE (I) were measured in peripheral blood mononuclear cells (PBMCs)of 8 NAR and 10 AR patients. Data shown are mean ± SD by an unpaired t-test; P1, patient of acute rejection post renal transplant; C1, control patient of stable kidney function post renal transplant; NAR, non-acute rejection; AR, acute rejection. [/fig] [fig] FIGURE 6 |FIGURE 7 |: Immune cells enrichment. Distribution of cell-type enrichment scores for AR and NAR. xCell, a bioinformatics tool, was used to provide an enrichment score for different cell types that allow comparison of cell types across group. The x-axis represents cell types. The y-axis represents the xCell enrichment score. Gene set enrichment analysis (GSEA). The full list of gene sets enriched in samples with DOCK2 (A), NCKAP1L (B), IL2RG (C), SLAMF8 (D), CD180 (E), and PTPRE (F) highly expressed. [/fig] [fig] FIGURE 8 |: SLAMF8 participate in AR progression via TLR4 in vivo. (A, B) Representative images and quantification of SLAMF8 + cells number in the human allograft diagnosed as TCMR (n=9) clinically and HC (n=11). Scale bar: 50 mm; six random fields were taken from each kidney. (C) Representative of immunofluorescence staining of TLR4 (green) and SLAMF8 (red) in TCMR (n=11) and HC (n=8). Red arrow indicates cells co-expressing TLR4 and SLAMF8. Scale bar = 20mm; TCMR, T-cell-mediated rejection; HC, healthy control. [/fig] [fig] FIGURE 9 |: SLAMF8 and TLR4 are co-expressed in M1-type macrophages. Relative mRNA expression of SLAMF8 (A) and TLR4 (B) were measured in M0, M1, and M2. (C-E) Representative and quantification of immunofluorescence staining of TLR4 (green) and SLAMF8 (red) in M0, M1, and M2. Red arrow indicates cells co-expressing TLR4 and SLAMF8. Scale bar = 50mm; six random fields were taken from each coverslip (mean ± SD, n = 6). (F) Representative of immunofluorescence staining of TLR4 (green) and SLAMF8 (red) in M1. Scale bar = 20mm. M0, murine RAW 264.7; M1, RAW 264.7 treated with 10 ng/ml LPS plus 20 ng/ml IFNg for 24 h; M2, RAW 264.7 treated with 10 ng/ml IL-4 for 24 h. [/fig]

Fungal Lactamases: Their Occurrence and Function # Introduction The soil is one of the most complex habitats on earth due primarily to the diversity of microorganisms that inhabit it and the myriad biochemical products they secrete. Experiments utilizing metagenomic technologies estimate up to several million species of bacteria per gram in some naturally occurring soils [bib_ref] Response to comment by Bunge et al. on "Computational improvements reveal great..., Gans [/bib_ref]. The majority of these species are, to date, uncultured. For fungi, less information is available. Earlier estimates suggested that there are approximately 1.5 million fungal species on the planet [bib_ref] The fungal dimension of biodiversity: magnitude, significance, and conservation, Hawksworth [/bib_ref] , but more recent global estimates of six million soil fungi were suggested based on comprehensive molecular studies [bib_ref] A first comprehensive census of fungi in soil reveals both hyperdiversity and..., Taylor [/bib_ref]. This microbial diversity creates a dynamic environment for microorganisms to communicate and compete for limited resources. Further, metabolic processes of microbes together with plants act as significant sources of chemical diversity, and microbes in the soil milieu are constantly and unavoidably exposed to foreign chemicals (xenobiotics). Some xenobiotics are easily tolerated and degraded, while others have inhibitory effects [bib_ref] Biotransformation of xenobiotics, Parkinson [/bib_ref]. Xenobiotics that are deleterious to the growth or metabolic activities of other microorganisms can be considered antibiotics, and play critical ecological roles in competitive interactions [bib_ref] Spatial variation in frequency and intensity of antibiotic interactions among streptomycetes from..., Davelos [/bib_ref] [bib_ref] Streptomyces competition and co-evolution in relation to plant disease suppression, Kinkel [/bib_ref]. It has been posited that microorganisms and plants have adopted antibiotic production as offensive and/or defensive strategies to adjust to changing circumstances, allowing microbial colonization in the rhizosphere or persistence of plants in the environment [bib_ref] Microbial diversity in soil: ecological theories, the contribution of molecular techniques and..., Lynch [/bib_ref]. Compared to the surrounding soil, the rhizosphere of plants can be particularly rich in nutrients [bib_ref] Development of specific rhizosphere bacterial communities in relation to plant species, nutrition..., Marschner [/bib_ref]. This microbial oasis stimulates competitive and antagonistic relationships among would-be colonizers. For example, phenazine production by pseudomonads and trifolitoxin production from certain Rhizobium species correlate with soil survival and suppressive activity, demonstrating that antibiotic production can be integral to niche competition and microbial community structure [bib_ref] Contribution of phenazine antibiotic biosynthesis to the ecological competence of fluorescent pseudomonads..., Mazzola [/bib_ref] [bib_ref] Effects of bacterial antibiotic production on rhizosphere microbial communities from a cultureindependent..., Robleto [/bib_ref]. Antibiotic production by both plants and microbes is a remarkable strategy possibly adopted in response to their sessile nature and limited mobility, respectively [bib_ref] Plant metabolic diversity: a regulatory perspective, Grotewold [/bib_ref] [bib_ref] Current trends in the evolutionary ecology of plant defence, Agrawal [/bib_ref]. Heritable genetic alterations such as mutation, gene duplication/modification, and horizontal gene transfer (HGT) have expanded the antibiotic repertoires of plants and microbes [bib_ref] Horizontal gene transfer: building the web of life, Soucy [/bib_ref]. A number of antibiotic families have been detected from soil or produced by soil microbes and display in vivo or in vitro antagonistic effects, such as penicillin, trichothecene, chloromycetin, actinomycin, clavacin, griseofulvin, etc. [bib_ref] Soil produced antibiotics-plant disease and insect control, Stallings [/bib_ref] [bib_ref] Rapid quantification of Bacillus subtilis antibiotics in the rhizosphere, Kinsella [/bib_ref]. Yet, antibiotics can occur in nature at sub-inhibitory concentrations, and rather than inhibiting growth, the compounds elicit transcriptional responses suggestive of a form of microbial communication [bib_ref] Transcriptional modulation of bacterial gene expression by subinhibitory concentrations of antibiotics, Goh [/bib_ref] [bib_ref] Are antibiotics naturally antibiotics?, Davies [/bib_ref]. In addition to microbial sources, compounds with antibiotic activity are also found in plants [bib_ref] Two classes of plant antibiotics: phytoalexins versus phytoanticipins, Vanetten [/bib_ref] [bib_ref] Oxazolidinones: activity, mode of action, and mechanism of resistance, Bozdogan [/bib_ref] [bib_ref] Plant antimicrobial agents and their effects on plant and human pathogens, González-Lamothe [/bib_ref]. Maackiain is a plantderived antibiotic extracted from red clover and alfalfa. Previous work has shown that maackiain is toxic to several genera of fungal pathogens of legume and non-legume hosts [bib_ref] Effect of treatment with the phytoalexins medicarpin and maackiain on fungal growth..., Duczek [/bib_ref] [bib_ref] Differential toxicity of enantiomers of maackiain and pisatin to phytopathogenic fungi, Delserone [/bib_ref]. Maize, wheat, and rye can constitutively produce benzoxazinones and benzoxazolinones, which help reduce insect damage and confer resistance to various fungal and bacterial pathogens [bib_ref] Role of cyclic hydroxamic acids in monogenic resistance of maize to Helminthosporium..., Couture [/bib_ref] [bib_ref] Antifungal compounds in winter wheat resistant and susceptible to Septoria nodorum, Baker [/bib_ref] [bib_ref] Two horizontally transferred xenobiotic resistance gene clusters associated with detoxification of benzoxazolinones..., Glenn [/bib_ref]. To combat antibiosis, bacteria have developed resistance mechanisms such as efflux pumps and hydrolytic enzymes. Notorious among the latter group, β-lactamases have been thoroughly studied due to the resistance they confer to the widespread clinically used β-lactam antibiotics. Parallel to the presence in bacteria, genes encoding "β-lactamases" are also abundant across different fungal families. In contrast to bacteria, almost nothing is known about the function of these genes in fungi. Previous work is limited to two studies on the hydrolytic function of lactamase (metallo-β-lactamase, MBL) encoding genes in Fusarium verticillioides and Fusarium pseudograminearum [bib_ref] A γ-lactamase from cereal infecting Fusarium spp. catalyses the first step in..., Kettle [/bib_ref] [bib_ref] Two horizontally transferred xenobiotic resistance gene clusters associated with detoxification of benzoxazolinones..., Glenn [/bib_ref]. This evidence serves as a foundational paradigm for studying hydrolytic lactamases in fungi and prompts the hypothesis that, as in bacteria, many of these enzymes function in degradation and resistance to xenobiotic compounds. In this review, we will describe an initial look at the distribution of lactamase-encoding genes in fungi and speculate on their ecological roles. We will also describe current and planned approaches to decipher the roles of 46 lactamase-family genes in the F. verticillioides genome. ## Lactams-the archetypical class of antibiotics Bactericidal β-Lactams β-Lactams comprise the largest group of antibiotics, and they have been extensively utilized for their antibacterial effect [bib_ref] Mode of action of beta-lactam antibiotics, Tipper [/bib_ref]. Beginning with Alexander Fleming's Nobel Prizewinning serendipitous discovery of a penicillin-producing mold, β-lactams and their semisynthetic derivatives have been the most impactful antibiotics in medicine [bib_ref] The β-lactam antibiotics: past, present, and future, Demain [/bib_ref] [bib_ref] Platforms for antibiotic discovery, Lewis [/bib_ref]. Their mode of action is well characterized and involves a four-membered cyclic amide ring [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref] that occupies the catalytic sites of transpeptidases, also referred to as penicillin-binding proteins. These proteins are essential for crosslinking peptidoglycan layers of bacterial cell walls, thus β-lactam antibiotics disrupt bacterial cell wall synthesis, resulting in cell lysis [bib_ref] Penicillin-binding proteins and the mechanism of action of beta-lactam antibiotics, Waxman [/bib_ref]. ## Lactam production in fungi Fungi are the original source of two foundational β-lactam antibiotics: penicillin and cephalosporin. These drugs are still industrially produced, primarily using Penicillium chrysogenum and Acremonium chrysogenum (previously Cephalosporium), respectively [bib_ref] Aspects on evolution of fungal β-lactam biosynthesis gene clusters and recruitment of..., Brakhage [/bib_ref]. Lactam production in fungi is frequently coordinated through the activity of gene clusters containing necessary biosynthetic enzymes and pathwayspecific transcriptional regulators [bib_ref] Aspects on evolution of fungal β-lactam biosynthesis gene clusters and recruitment of..., Brakhage [/bib_ref] [bib_ref] SMURF: genomic mapping of fungal secondary metabolite clusters, Khaldi [/bib_ref] [bib_ref] Secondary metabolic gene clusters: evolutionary toolkits for chemical innovation, Osbourn [/bib_ref] [bib_ref] Fungal secondary metabolitesstrategies to activate silent gene clusters, Brakhage [/bib_ref]. Fungal gene clusters are hypothesized to assist in retention of biochemical functions by reducing gene loss due to recombination in highly dynamic genomes [bib_ref] Secondary metabolic gene clusters: evolutionary toolkits for chemical innovation, Osbourn [/bib_ref]. Fungal genomes provide enormous potential to produce many complex lactam-containing compounds [fig_ref] FIGURE 2 |: Examples of lactam-containing fungal compounds [/fig_ref] , including higher order lactam compounds (e.g., five-membered, γ-lactam rings). Two new hetero-spirocyclic γ-lactams, azaspirofurans A and B, were isolated from a marine sediment-derived fungus Aspergillus sydowii [bib_ref] Two new hetero-spirocyclic γ-lactam derivatives from marine sediment-derived fungus Aspergillus sydowi D2-6, Ren [/bib_ref]. A maize seed-borne endophyte Sarocladium zeae (formerly Acremonium zeae) was found to produce γ-lactam compounds, named pyrrocidine A and B [bib_ref] Pyrrocidines A and B, new antibiotics produced by a filamentous fungus, He [/bib_ref]. Further, the cytotoxic awajanomycin from Acremonium species, cytochalasins from Rhinocladiella, and colletotrilactams A-D from endophytic Colletotrichum gloeosporioides all exemplify fungal production of higher order lactams [bib_ref] Three new cytochalasins produced by an endophytic fungus in the genus Rhinocladiella, Wagenaar [/bib_ref] [bib_ref] Awajanomycin, a cytotoxic gamma-lactone-delta-lactam metabolite from marine-derived Acremonium sp. AWA16-1, Jang [/bib_ref]. Such lactam production among fungi diversifies xenobiotic composition in soil and may contribute to the discovery of new valuable antibiotics. ## Antifungal lactams In addition to the fungal production of bactericidal lactams, emerging evidence indicates that certain atypical lactams can be fungistatic or fungicidal regardless of their origins [fig_ref] FIGURE 3 |: Fungicidal or fungistatic lactams [/fig_ref] ; [bib_ref] Aspects on evolution of fungal β-lactam biosynthesis gene clusters and recruitment of..., Brakhage [/bib_ref]. Novel monocyclic N-thiolated β-lactams revealed varying degrees of in vitro antifungal activity against seven Candida species [bib_ref] Studies on the antifungal properties of N-thiolated beta-lactams, O&apos;driscoll [/bib_ref]. The fungistatic mode of action against Candida was postulated to simulate what was observed against Staphylococcus aureus, where these lactams diffused through the cell membrane and interacted covalently with an unknown and possibly evolutionarily conserved target. Two synthetic azetidin-2-one compounds showed moderate antifungal activity against Botrytis cinerea, Colletotrichum lindemuthianum, and the oomycete Phytophthora infestans [bib_ref] Activity of a series of beta-lactams against some phytopathogenic fungi, Arnoldi [/bib_ref]. The previously mentioned pyrrocidine A and B from S. zeae are antagonistic to kernel rotting fungi including Aspergillus flavus and F. verticillioides [bib_ref] A protective endophyte of maize: Acremonium zeae antibiotics inhibitory to Aspergillus flavus..., Wicklow [/bib_ref]. Interestingly, pyrrocidine A differs from B only in that it possesses a double bond in the γ-lactam ring, and pyrrocidine A shows inhibition at a lower concentration than does B, implying the relevance of the lactam ring to antibiosis. Alternatively, structural conformation changes conveyed by the single vs. double bond could potentially play a role in the observed differential toxicity. Recent studies on synthetic bicyclic lactam analogs of natural plant derived lactones have also revealed their fungistatic effects against B. cinerea, Penicillium citrinum, and Aspergillus glaucus [bib_ref] Synthesis and fungistatic activity of bicyclic lactones and lactams against Botrytis cinerea,..., Walczak [/bib_ref]. For example, by replacing an oxygen atom with nitrogen in the fivemembered ring during a heteroatom analysis of cis-3-oxabicyclo-[4.3.0]non-7-en-2-one, a novel γ-lactam compound was created with a significant increase in antifungal activity [bib_ref] Synthesis and fungistatic activity of bicyclic lactones and lactams against Botrytis cinerea,..., Walczak [/bib_ref]. These discoveries should stimulate further exploration of antifungal lactams and their modes of action. ## Β-lactamases ## Lactam resistance The spread of antibiotic resistance among bacteria is one of today's major world health concerns [bib_ref] Tackling antibiotic resistance: the environmental framework, Berendonk [/bib_ref]. In fact, many current publications in the popular press are predicting the end of the age of antibiotics in the near future [bib_ref] The Superbug that Doctors have been Dreading just Reached the U, Sun [/bib_ref] , and the World Health Organization recently held a conference on the subject entitled "The end of antibiotics?" Natural sources and clinical/agricultural overuse of antibiotics impose selection pressure for antibiotic resistance, leading to a rise in the number of resistant microbes and the spread of resistant genes regardless of their origins [bib_ref] Call of the wild: antibiotic resistance genes in natural environments, Allen [/bib_ref] [bib_ref] Antibiotics in agriculture and the risk to human health: how worried should..., Chang [/bib_ref]. Currently, three major mechanisms have been proposed to generate resistance to β-lactam antibiotics : (1) restricted access to drug targets either by (a) preventing drug entry or (b) enhanced drug efflux [bib_ref] Role of efflux pump(s) in intrinsic resistance of Pseudomonas aeruginosa: active efflux..., Li [/bib_ref] , (2) alteration of drug targets [bib_ref] Modification of penicillin-binding proteins as mechanisms of beta-lactam resistance, Malouin [/bib_ref] , or (3) the presence of drug-degrading enzymes [bib_ref] β-Lactams: chemical structure, mode of action and mechanisms of resistance, Fernandes [/bib_ref]. Moderate lactam resistance may be developed by intragenic recombination, where genetically distinct alleles occasionally are produced. Such events generate, for example, new alleles of mosaic transpeptidase (penicillin target protein) genes with low penicillin-binding affinities [bib_ref] Genetic diversity of penicillin-binding protein 2 genes of penicillinresistant strains of Neisseria..., Zhang [/bib_ref] [bib_ref] Genetic diversity of penicillin-resistant Neisseria meningitidis, Campos [/bib_ref]. HGT was proposed decades ago as another means of acquisition of lactam resistance. HGT appears responsible for the spread of both resistance-conferring transpeptidases and (1) Penicillin, the historically significant fungal lactam produced by Penicillium chrysogenum [bib_ref] On the antibacterial action of cultures of a Penicillium, with special reference..., Fleming [/bib_ref] ; (2) cephalosporins, a group of bactericidal β-lactams from Acremonium chrysogenum [bib_ref] Cephalosporins: a review, Harrison [/bib_ref] ; (3) gliotoxin, a mycotoxin produced by Aspergillus fumigatus and several other species [bib_ref] Identification of cryptic products of the gliotoxin gene cluster using NMR-based comparative..., Forseth [/bib_ref] ; (4) sirodesmin PL, a phytotoxin produced by the fungus Leptosphaeria maculans causing blackleg disease of canola [bib_ref] The sirodesmin biosynthetic gene cluster of the plant pathogenic fungus Leptosphaeria maculans, Gardiner [/bib_ref] ; (5) roquefortine C, a mycotoxin produced by Penicillium species [bib_ref] The effect of substrate on mycotoxin production of selected Penicillium strains, Kokkonen [/bib_ref] ; (6) meleagrin, a bioactive alkaloid produced by deep ocean Penicillium [bib_ref] Isolation of radicicol from Penicillium luteoaurantium, and meleagrin, a new metabolite, from..., Nozawa [/bib_ref] ; (7) cyclopiazonic acid, a toxic fungal secondary metabolite originally isolated from Penicillium cyclopium [bib_ref] The isolation and structure of cyclopiazonic acid a toxic metabolite of Penicillium..., Holzapfel [/bib_ref] ; (8) equisetin, a Fusarium equiseti metabolite [bib_ref] Isolation and characterization of novel human immunodeficiency virus integrase inhibitors from fungal..., Hazuda [/bib_ref] ; (9) ilicicolin H is an NRPS-polyketide hybrid product discovered from Cylindrocladium iliciola MFC-870 and is a potent antifungal agent [bib_ref] Biotransformation of antifungal ilicicolin H, Singh [/bib_ref] ; (10) ergotamine, an ergopeptine and part of the ergot family of alkaloids from Claviceps purpurea [bib_ref] Ergot and its alkaloids, Schiff [/bib_ref] ; (11) cytochalasin D, a cytostatically active metabolite isolated from Tubercularia species [bib_ref] X-ray crystal structure of cytochalasin D produced by Tubercularia sp., a novel..., Wang [/bib_ref] ; (12) cytochalasin E from Rhinocladiella species [bib_ref] Three new cytochalasins produced by an endophytic fungus in the genus Rhinocladiella, Wagenaar [/bib_ref] ; (13) azaspirofuran A and (14) azaspirofuran B produced by Aspergillus sydowii [bib_ref] Two new hetero-spirocyclic γ-lactam derivatives from marine sediment-derived fungus Aspergillus sydowi D2-6, Ren [/bib_ref] ; (15) awajanomycin produced by Acremonium species [bib_ref] Awajanomycin, a cytotoxic gamma-lactone-delta-lactam metabolite from marine-derived Acremonium sp. AWA16-1, Jang [/bib_ref] ; (16) fusarin C, a mycotoxin produced by several Fusarium species [bib_ref] Fusarin C, a mutagen from Fusarium moniliforme grown on corn, Wiebe [/bib_ref]. plasmid-encoded β-lactamases contributing to high-level lactam resistance and the appearance of "superbugs" with resistance to most or all current antibiotic therapies [bib_ref] Penicillin-resistant Viridans streptococci have obtained altered penicillin-binding protein genes from penicillin-resistant strains..., Dowson [/bib_ref] [bib_ref] Horizontal spread of an altered penicillin-binding protein 2B gene between Streptococcus pneumoniae..., Coffey [/bib_ref] [bib_ref] Integrons and beta-lactamases -A novel perspective on resistance, Weldhagen [/bib_ref] [bib_ref] Origins and evolution of antibiotic resistance. Microbiol, Davies [/bib_ref]. Bacterial β-Lactamases β-Lactamase enzymes are the most common mechanism of resistance to β-lactam antibiotics, hydrolyzing the lactam bond in their four-membered ring structures to abolish activity [bib_ref] Beta-lactamase-mediated resistance and opportunities for its control, Livermore [/bib_ref]. As these antibiotics are classically active against peptidoglycan cell wall synthesis, the corresponding hydrolytic β-lactamases result in high prevalence of resistant strains and a potential increase in virulence. The first penicillin-hydrolyzing β-lactamase identified was an AmpC cephalosporinase in Escherichia coli in 1940, several years before the actual introduction of penicillin into clinical practice [bib_ref] An enzyme from bacteria able to destroy penicillin, Abraham [/bib_ref]. Two primary schemes of classifying bacterial β-lactamases have been proposed based on functionality or molecular characteristics [fig_ref] TABLE 1 |: Classification of bacterial β-lactamases [/fig_ref]. The functionality classification scheme divides bacterial β-lactamases into three major groups based on inhibitory specificities and the potential requirement of zinc ion for activity [bib_ref] Beta-lactamases and bacterial resistance to antibiotics, Frère [/bib_ref]. Group 1 includes cephalosporinases that are not well inhibited by active sitedirected β-lactamase inhibitors, such as clavulanic acid. Group 2 encompasses β-lactamases that are inhibited by clavulanic acid. Group 3 refers to MBLs that require zinc ions for activity. In addition to conventional hydrolases targeting β-lactams, the MBL superfamily includes lactonases that hydrolyze lactone bonds. A classic example is N-acyl homoserine lactonase produced by various bacteria. These lactonases are able to inactivate (3) (±)-cis-3-azabicyclo[4.3.0]non-7-en-2-one, an artificially synthesized compound that is also fungistatic to the same three species as Vince lactam [bib_ref] Synthesis and fungistatic activity of bicyclic lactones and lactams against Botrytis cinerea,..., Walczak [/bib_ref] ; (4) 2-chloromethyl-3-methyl-4(3H)-quinazolinone, exhibiting antifungal activity against Fusarium oxysporum and Macrophomina sorgina [bib_ref] Antibacterial, antifungal and antifeedant activity of quinazolinonyl-β-lactams/quinazolinones and bis (quinazolinonyl-β-lactams), Reddy [/bib_ref] ; (5) (±)-2-butyl-2-azabicyclo[2.2.1]hept-5-en-3-one, an artificially synthesized compound that moderately inhibits the growth of A. glaucus; (6) 1,2,3-triazole-linked β-lactam-bile acid conjugates (R 1 = H or Cl, R 2 = H or OH), a group of artificially synthesized compounds that inhibit the growth of F. oxysporum, Candida albicans, Cryptococcus neoformans, Benjaminiella poitrasii, Yarrowia lipolytica [bib_ref] Synthesis and antimicrobial activity of beta-lactambile acid conjugates linked via triazole, Vatmurge [/bib_ref] ; (7) maltophilin, produced by a ubiquitous free-living bacterium Stenotrophomonas maltophilia, which demonstrates inhibitory effects against several Ascomycetes, such as Aspergillus terreus, B. cinerea, C. albicans, Fusarium solani, etc. [bib_ref] Maltophilin: a new antifungal compound produced by Stenotrophomonas maltophilia R3089, Jakobi [/bib_ref] ; (8) flucytosine, an effective antifungal compound indicated for the treatment of serious infections caused by susceptible strains of Candida or Cryptococcus neoformans [bib_ref] Flucytosine primary resistance in Candida species and Cryptococcus neoformans, Cuenca-Estrella [/bib_ref] ; (9, 10) pyrrocidine A and B, respectively, broad spectrum antibiotics produced by Sarocladium zeae [bib_ref] Pyrrocidines A and B, new antibiotics produced by a filamentous fungus, He [/bib_ref]. N-acyl homoserine lactones by hydrolyzing the lactone bond, resulting in quenching of bacterial quorum-sensing signaling [bib_ref] Quenching quorum-sensing-dependent bacterial infection by an N-acyl homoserine lactonase, Dong [/bib_ref] [bib_ref] A metagenomic analysis of soil bacteria extends the diversity of quorumquenching lactonases, Riaz [/bib_ref]. The necessity of zinc ions is suspected by the universal presence of a conserved dinuclear zinc binding site in known lactonases and confirmed by zinc's essential role during catalytic activity and protein folding [bib_ref] The quorum-quenching lactonase from Bacillus thuringiensis is a metalloprotein, Thomas [/bib_ref]. The second scheme for classification of β-lactamases utilizes nucleotide and amino acid sequences to divide them into four molecular classes designated A-D. Enzymes belonging to class A, C, and D act by a serine-based mechanism, often containing Pfam domain PF00144. Those in class B are zinc-based MBLs with Pfam domain PF00753, equivalent to functional Group 3. Serine-based β-lactamases (SBLs) possess conserved motifs S-X-X-K, S/Y-X-N/V, and K-T/S-G in that order, where the serine in the first motif serves as the active site targeting the β-lactam ring. Class B β-lactamases contain a primary zinc-binding motif H-X-H-X-D-H followed by conserved amino acids of Gly, Leu, His, Gly, Asn, and His at specific positions. Except for these conserved amino acids, the rest of their sequences are generally divergent, with greatly differing tertiary structures and catalytic efficiencies [bib_ref] Avibactam is a covalent, reversible, non-β-lactam β-lactamase inhibitor, Ehmann [/bib_ref]. ## Fungal lactamases Interestingly, genes encoding proteins with β-lactamase homology are widely distributed across major taxa. As of March 1, 2017, there were 1,096,469 manually and computationally annotated β-lactamase encoding genes reported in the National Center for Biotechnology Information (NCBI) protein database across all kingdoms of life. As depicted in , 93% of them (1,021,177 genes) lie in the domain Bacteria. Although non-bacterial lactamases share similarities with those found in bacteria, less than 1% have been functionally characterized. It is very likely that many non-bacterial "β-lactamases" are not involved in degrading classic β-lactams, so we will refer to them simply as lactamases below. Interestingly, of the FIGURE 4 | Three major resistant mechanisms present in bacteria against β-lactam antibiotics. Bacteria can develop resistance to β-lactams by (1) restricting their access to penicillin binding proteins; (2) altering penicillin binding proteins to avoid being recognized by β-lactams; (3) producing β-lactamases. Generally speaking, Gram-negative bacteria retain β-lactamases in the periplasmic space between inner and outer membranes. In contrast, Gram-positive bacteria do not possess an outer membrane, and they usually release β-lactamases to the extracellular environment. Fine details of the bacterial membranes and peptidoglycan layer are not shown in this simplified drawing. roughly one million database entries with suspected lactamase homologs, 14,923 genes were found in fungi, which represents approximately half of the eukaryotic total (29,804 genes). Due to ever-increasing affordability and ease of sequencing, newly identified genes encoding putative lactamases are being added at an accelerating rate to databases. Even though a large number of fungal genes have been identified that putatively encode lactamases with Pfam domains PF00144 or PF00753 similar to bacteria, only a few gene products have confirmed functions. For example, Saccharomyces cerevisiae possesses a small core set of highly conserved enzymes with lactamase domains, but they tend to have specialized functions not involving lactam hydrolysis [fig_ref] TABLE 2 |: Saccharomyces cerevisiae lactamase orthologs in three Fusarium species [/fig_ref]. The essential gene TRZ1 from S. cerevisiae encodes tRNase Z, involved in RNA processing [bib_ref] Characterization of TRZ1, a yeast homolog of the human candidate prostate cancer..., Chen [/bib_ref] [bib_ref] The role of the Brr5/Ysh1 C-terminal domain and its homolog Syc1 in..., Zhelkovsky [/bib_ref]. The essential endonuclease YSH1 in S. cerevisiae contains a MBL domain and plays key roles in pre-mRNA 3 end formation, cooperating with other cleavage factors (Stumpf and Domdey, FIGURE 5 | Sunburst visualization of β-lactamase gene distribution by major taxon. Each node of the taxonomic hierarchy is represented as a separate arc, arranged radially with the domains at the center and the phyla arrayed around the outermost ring. The area of each arc is proportional to the number of β-lactamases reported in the NCBI protein database. I, M, and O refer to inner, middle, and outer arcs. The frequency of Fusarium lactamases among the Ascomycota is denoted in bar chart form. [fig_ref] TABLE 2 |: Saccharomyces cerevisiae lactamase orthologs in three Fusarium species [/fig_ref] , possess YSH1 and TRZ1 orthologs in their genomes. Non-essential fungal lactamases appear to have diversified functions, not restricted to nucleases. The non-essential BDS1 in S. cerevisiae, presumably horizontally acquired from bacteria, possesses an MBL domain and functions as a sulfuric ester hydrolase [bib_ref] Contribution of horizontal gene transfer to the evolution of Saccharomyces cerevisiae, Hall [/bib_ref]. A discrete MBL type thioesterase in Aspergillus fumigatus was found to be required for biosynthesis of endocrocin, a simple anthraquinone commonly identified in fungal extracts [bib_ref] Genomebased cluster deletion reveals an endocrocin biosynthetic pathway in Aspergillus fumigatus, Lim [/bib_ref]. Asperthecin, a polyketide anthraquinone pigment, is produced by certain Aspergillus species [bib_ref] Studies in the biochemistry of micro-organisms. 94. The colouring matters of species..., Howard [/bib_ref] , and disruption of the asperthecin biosynthetic gene cluster in Aspergillus nidulans revealed that a lactamase assisted the adjacent polyketide synthase to hydrolyze an aromatic polyketide into endocrocin-9-anthrone [bib_ref] Identification and characterization of the asperthecin gene cluster of Aspergillus nidulans, Szewczyk [/bib_ref]. LovD, a SBL containing the PF00144 motif, in Aspergillus terreus was essential for lovastatin biosynthesis, and it was also later described to be involved in synthesizing simvastatin, a lipid-lowering agent, by acting on a protein-bound acyl substrate [bib_ref] Modulation of polyketide synthase activity by accessory proteins during lovastatin biosynthesis, Kennedy [/bib_ref] [bib_ref] The role of distant mutations and allosteric regulation on LovD active site..., Jiménez-Osés [/bib_ref]. Through proteomic studies on both weakly and highly aggressive Verticillium dahliae isolates, it was inferred that a β-lactamase family protein might act as a pathogenicity factor that is recognized by the host plant immune system as an elicitor [bib_ref] Proteomic analysis of the phytopathogenic soilborne fungus Verticillium dahliae reveals differential protein..., El-Bebany [/bib_ref]. Thus, the functional diversity of fungal lactamases is evident despite limited studies. ## 1996). filamentous fungi, including fusarium Recent literature has shown that two related fungal lactamases function in xenobiotic hydrolysis, similar to bacterial counterparts. In fact, our interest in fungal lactamases stems from the observation that the gene FVEG_08291 in F. verticillioides encodes a lactamase designated MBL1 that is responsible for the degradation of 2-benzoxazolinone (BOA) [bib_ref] Two horizontally transferred xenobiotic resistance gene clusters associated with detoxification of benzoxazolinones..., Glenn [/bib_ref]. A similar but non-orthologous MBL in F. pseudograminearum (FPSE_08124) was also shown to be responsible for BOA degradation [bib_ref] Degradation of the benzoxazolinone class of phytoalexins is important for virulence of..., Kettle [/bib_ref]. BOA is a γ-lactam phytochemical produced by select graminaceous crops that is implicated in resistance to insect herbivory and microbial pathogens. The enzymatic capacity of Fusarium species to hydrolyze BOA is suggested to enhance colonization of the host, thus increasing the frequency and abundance of the species [bib_ref] Host-synthesized secondary compounds influence the in vitro interactions between fungal endophytes of..., Saunders [/bib_ref] [bib_ref] Exploring the evolutionary ecology of fungal endophytes in agricultural systems: using functional..., Saunders [/bib_ref]. Interestingly, MBL1 is part of a gene cluster that is up-regulated in response to BOA, and this cluster, called the FDB1 cluster, was also observed in the other maize pathogens Fusarium subglutinans and Colletotrichum graminicola [bib_ref] Two horizontally transferred xenobiotic resistance gene clusters associated with detoxification of benzoxazolinones..., Glenn [/bib_ref]. The highly conserved synteny of the FDB1 cluster between these fungi suggests C. graminicola acquired the cluster from Fusarium by HGT, and that the maize host and its phytochemicals, notably BOA and related lactams, are driving factors influencing the evolution and genomic content of these fungi. ## Fusarium lactamases Fusarium Lactamase Analysis as a Paradigm? Our analysis suggests that soil-borne fungi tend to possess more lactamase encoding genes compared with the minimal sets from fungi predicted to live in environments of relatively low microbial diversity [fig_ref] FIGURE 6 |: Lactamase genes are abundant within some fungi [/fig_ref]. Broadly distributed in soil, Fusarium species are likely in competition with diverse microbes and are presumably often exposed to xenobiotic compounds. Frequent confrontation with competing microorganisms inhabiting overlapping ecological niches is expected to hone genetic determinants of xenobiotic resistance. Fusarium interactions with soil competitors are complex, involving nutrient competition and chemical warfare [bib_ref] Streptomyces competition and co-evolution in relation to plant disease suppression, Kinkel [/bib_ref]. As noted in , the majority (84.3%) of sequenced fungal lactamase encoding genes are from the phylum Ascomycota (12,771 genes), 11.6% of which belonged to the genus Fusarium (1479 genes). There were, on average, 37 lactamase encoding genes per Fusarium species, as opposed to 15 per species among non-Fusarium fungal genomes. The species noted with the highest number of lactamase encoding genes, 88, was the soil limited root pathogen Fusarium solani [fig_ref] FIGURE 6 |: Lactamase genes are abundant within some fungi [/fig_ref]. Thus, we further propose that the abundance of Fusarium lactamases is likely integral to the success of this genus as a soil competitor. Further analysis of the global and individual roles of lactamases is important for more fully understanding Fusarium biology and its ecological interactions. ## Detailed analysis of f. verticillioides lactamases To better understand molecular characteristics of Fusarium lactamases, we cataloged the complete set of lactamase encoding genes from three representative and pathogenically important sequenced Fusarium genomes, F. verticillioides 7600 (Fv), Fusarium oxysporum 4287 (Fo), and Fusarium graminearum PH-1 (Fg), via homology-based protein reciprocal BLAST and bacterial β-lactamase HMMER sequence logo scanning [bib_ref] Nhmmer: DNA homology search with profile HMMs, Wheeler [/bib_ref]. We identified 46 lactamase domain-containing genes in Fv, 63 in Fo, and 38 in Fg, as listed in [fig_ref] TABLE 3 |: β-lactamase domain-containing genes in three Fusarium genomes [/fig_ref] by predicted enzymatic mechanisms (MBLs and SBLs). PSI-BLAST of each lactamase encoding gene in F. verticillioides helped uncover distant homologs and confirm domain integrity. Interestingly, some predicted SBL gene annotations (FVEG_03300, FVEG_14143, FVEG_15166, FVEG_17257, FVEG_17258) were missing core catalytic serine motifs or possessed only part of the conventional β-lactamase folds. Thus, these five genes were further evaluated for their open reading frames using the FGENESH program from Softberry (http://www.softberry.com) to refine gene predictions. Reannotated sequences suggested FVEG_17257 and FVEG_17258 should be merged as one lactamase encoding gene, while FVEG_03300, FVEG_14143, and FVEG_15166 remained unchanged, still missing the core serine and lacking canonical amino acids at the majority of conserved sites. These three were thus excluded from later syntenic and phylogenetic analyses. PSI-BLAST of MBLs in F. verticillioides also predicted several members could be involved in metabolizing RNA (FVEG_05485, FVEG_11466, FVEG_14723), degrading lipids (FVEG_11923, FVEG_03849), MBL, metallo-β-lactamases; SBL, serine-based β-lactamases. Bolded accession numbers are those with signal peptides identified by SignalP 4.1 Server, suggesting likely secretion [bib_ref] SignalP 4.0: discriminating signal peptides from transmembrane regions, Petersen [/bib_ref]. Amino acid sequences of F. verticillioides β-lactamases sharing more than 40% sequence identity are considered as paralogs and grouped in outlined boxes. (A) MBLs synteny. (B) SBLs synteny. The modified nucleotide sequence merging FVEG_17257 and FVEG_17258 based on FGENESH prediction was renamed FVEG_17257 * here and used for syntenic studies. (C) Demonstration of synteny of genes flanking β-lactamases exemplified by FVEG_12637, which represents part of the FDB2 gene cluster essential for the biotransformation of 2-benzoxazolinone [bib_ref] FDB2 encodes a member of the arylamine N-acetyltransferase family and is necessary..., Glenn [/bib_ref] [bib_ref] Two horizontally transferred xenobiotic resistance gene clusters associated with detoxification of benzoxazolinones..., Glenn [/bib_ref]. Orthologs are shown in the same color with accession numbers above, and direction of arrows represents the orientation of genes. repairing DNA (FVEG_00815, FVEG_04252), and hydrolyzing hydroxylacyl glutathione (FVEG_08018, FVEG_16907), which also require zinc ions for appropriate functions. See also [fig_ref] TABLE 2 |: Saccharomyces cerevisiae lactamase orthologs in three Fusarium species [/fig_ref]. Fv-oriented syntenic studies were performed such that corresponding orthologs and adjacent genes in Fg and Fo were examined. In terms of species phylogeny, Fv is more closely related to Fo than Fg. Thus, we naturally expected more orthologs identified in Fo. Except for those Fv lactamase encoding genes with no orthologs in the other two species, the rest of the genes generally retained syntenic clusters in the Fg and/or Fo genomes [fig_ref] FIGURE 7 |: Orthological and syntenic analysis of Fusarium verticillioides, Fusarium graminearum, and F [/fig_ref]. Phylogenetic evaluation of 41 Fv genes having the core lactamase motifs revealed a high consistency with species evolution , where 37 genes fall into position A, clustering with orthologs in Fusarium fujikuroi in the respective phylograms. This suggests that Fv lactamases are most similar to those annotated in closely related species compared with other relatively distant species. Lactamase encoding genes in Fusarium species generally form a clade distinct from other Sordariomycetes. Interestingly, only 29% of the Fv MBLs had evidence of paralogy [fig_ref] FIGURE 7 |: Orthological and syntenic analysis of Fusarium verticillioides, Fusarium graminearum, and F [/fig_ref] , whereas 63% of the Fv SBLs appeared to have paralogs. This suggests the two types of lactamases may have different evolutionary pressures impacting duplication and diversification. Only six Fv lactamase encoding genes lack possible orthologs in both Fo and Fg [fig_ref] FIGURE 7 |: Orthological and syntenic analysis of Fusarium verticillioides, Fusarium graminearum, and F [/fig_ref]. Collectively the data indicate that some lactamase encoding genes originated before the divergence of Fusarium species, resulting in greater sequence diversity accompanying species divergence. Interestingly, FVEG_12347 was the only gene in the phylogenetic position B , suggesting that it is similar to Fg ortholog FGSG_04727 and lacks an ortholog in Fo [fig_ref] FIGURE 7 |: Orthological and syntenic analysis of Fusarium verticillioides, Fusarium graminearum, and F [/fig_ref]. FVEG_08291, FVEG_09433, and FVEG_12457 notably exhibited more similarities to orthologs in other Sordariomycetes rather than in closely related Fusarium species. These fall into phylogenetic position C and are thus good candidates for HGT derivation. The FVEG_08291 protein sequence possessed 85% identity to an ortholog in C. graminicola (NCBI Reference Sequence: XP_008099767.1), another Sordariomycetes pathogen FIGURE 8 | Phylogenetic placement of predicted Fusarium verticillioides lactamase genes based on amino acid sequence alignment. This cartoon summarizes the phylogenetic pattern of lactamase genes with intact core motifs in F. verticillioides. Each query lactamase amino acid sequence was searched for its top 50 homologs using BLASTP in NCBI. Neighbor-joining trees using the Jukes-Cantor genetic distance model were constructed by Geneious Tree Builder (Ver. 8.1) for each individual homology search. All tree topographies complied with the configuration such that each F. verticillioides lactamase fell into one of three positions marked as A, B, and C. Collapsed clades are shown as triangles. FIGURE 9 | Sequence alignment of motifs within Fusarium verticillioides metallo-β-lactamase proteins that are presumably associated with lactam hydrolysis. Amino acids matching at least 50% of all sequences are highlighted. of maize, surpassing homology to other related genes in Fusarium species. This is the MBL1 gene noted above as part of the FDB1 cluster conferring resistance to BOA. A similar case was observed for FVEG_09433, where it was more closely related to orthologs in C. graminicola and other genera than to those of most other Fusarium species, even though there are apparent FIGURE 10 | Sequence alignment of motifs within Fusarium verticillioides serine-based β-lactamases proteins that are presumably associated with lactam hydrolysis. Amino acids matching at least 75% of all sequences included were highlighted. orthologs in Fusarium mangiferae (GenBank ID: CVL02248.1) and F. fujikuroi (GenBank ID: CCT69225.1). One possible explanation is that FVEG_09433 was introduced to Fusarium species within the F. fujikuroi species complex prior to the divergence of these three species, but its orthologs among other species of the complex were somehow lost. The serine-based lactamase encoded by FVEG_12457 was most similar to its orthologs in A. terreus (NCBI Reference ID: XP_001217058.1) and Penicillium roqueforti (GenBank ID: CDM29397.1) with a sequence identity of over 70%, exceeding the 60% average identity among related Fusarium genes. Multiple Alignment using Fast Fourier Transform (MAFFT) analysis of presumed hydrolysis-related lactamase protein sequences was performed separately for MBLs and SBLs, presenting two distinctive patterns of conserved motifs [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref]. Although these Fv lactamases exhibited considerable sequence diversity, conserved motifs were still observed. As to MBLs, the conserved motif His-X-His-X-Asp-His-X-Gly resembled that in classic bacterial MBLs . However, compared to typical cases in bacteria, the overall conserved motif pattern is different in Fv MBLs, and additional motifs were identified, including Pro-X-Gly-His in Motif 3, Gly-Asp in Motif 4, and Pro-Gly in Motif 5 . A retrospective scrutiny of conserved motifs of bacterial PSI-BLAST hits revealed that these sites in Fv lactamases are also present in certain bacterial β-lactamases (data not shown). Fv SBLs demonstrate an interesting molecular pattern that is not present in bacteria [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref]. A total of nine motifs were identified in all intact Fv SBLs that are predicted to be hydrolysisassociated. Besides the catalytic core motif shared with bacteria (Ser-X-X-Lys as Motif 1), Fv lactamases contain conserved amino acids Leu-X-X-X-Gly in Motif 2, Pro-Glu-Leu in Motif 3, Leu-X-X-His-X-X-Gly in Motif 4, Pro-X-X-X-X-X-X-Tyr in Motif 5, Glu-X-X-X-Gly in Motif 6, a single conserved amino acid Pro in Motif 7, the Asp in Motif 8, and the Leu in Motif 9. However, none of these motifs (Motifs 2-9) are represented in bacterial species. Phyre2 predictions of tertiary structures reflected an interesting discovery that the majority of Fv MBLs were similar to bacterial β-lactamases, presenting a α-β/β-α sandwich structure composed of two β sheets at the core and α helices on the external surfaces [bib_ref] The Phyre2 web portal for protein modeling, prediction and analysis, Kelley [/bib_ref]. Those conserved residues are generally located at flexible loops connecting different secondary structures. It can be inferred that the spatial adjacency of histidines would facilitate the coordination of zinc ions and that the aspartic acid residues participate in the hydrolysis reaction. As exemplified in [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref] , FVEG_08291 was predicted to have the signature sandwich conformation with a flap structure (the flexible mobile loop), which is situated at the bottom of a wide shallow groove between two β-sheets [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref]. This structure has proven to be critical in substrate binding in bacteria [bib_ref] Identification of residues critical for metallobeta-lactamase function by codon randomization and selection, Materon [/bib_ref]. Superimposition of protein structures revealed that FVEG_08291 resembles a quorum-quenching lactonase (AiiB) from Agrobacterium tumefaciens [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref]. The conserved zinc-coordinated residues on the flexible loop as well as the easily accessible groove placement suggest the potential to accommodate various lactam or lactone molecules [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref]. Other conserved amino acids not directly predicted to be associated with catalytic reactions may be involved in structure maintenance or substrate recognition, and overall the catalytic mechanisms of fungal lactamases require further exploration. ## Conclusion and future directions The complexity of soil environments, particularly those with nutrient-driven competition in the rhizosphere, has led to diverse organisms capable of antimicrobial activity. Plants also contribute to rhizospheric antimicrobial content, either proactively (phytoanticipins), or reactive to pathogen contact (phytoalexins) [bib_ref] Fungal resistance to plant antibiotics as a mechanism of pathogenesis. Microbiol, Morrissey [/bib_ref] [bib_ref] Secretion of momilactone A from rice roots to the rhizosphere, Kato-Noguchi [/bib_ref]. Thus, the soil environment contains high antibiotic diversity including β-lactams, tetracyclines, sulfonamides, aminoglycosides, imidazoles, etc. [bib_ref] Pharmaceutical antibiotic compounds in soils -A review, Thiele-Bruhn [/bib_ref]. Competitive relationships among soil microflora exert FIGURE 12 | Potential ecological sources of lactam compounds and lactamases. Soil fungi, bacteria, plants, and protective endophytes (e.g., Sarocladium zeae) have all been documented to produce lactam-containing compounds. The antibiotic characteristic of these lactams has been implied to be associated with ecological niche competition. Plants, bacteria, fungi, and even animals have developed corresponding hydrolytic lactamases to combat antibiosis. To expand the conventional examples of bacterial β-lactamases, we propose a more generic ecological model linking lactam production with possible hydrolytic functions of organismal lactamases given their universal presence across different kingdoms. Note that arrow width depicts predicted relative abundance based on genomic frequencies. selective pressure on genes for antibiotic production and resistance. These genes in turn shape microbial populations and diversity, largely through development of antibiotic resistance mechanisms, such as the enzymatic degradation of β-lactamcontaining compounds. Xenobiotic degradation in soil is propelled by enzymatic processes such as hydrolysis, oxidative decarboxylation, and hydroxylation [bib_ref] Microbial models of soil metabolism: biotransformations of danofloxacin, Chen [/bib_ref] [bib_ref] Biodegradation of phosphonomycin by Rhizobium huakuii PMY1, Mcgrath [/bib_ref] [bib_ref] Biodegradability of cefotiam, ciprofloxacin, meropenem, penicillin G, and sulfamethoxazole and inhibition of..., Al-Ahmad [/bib_ref] [bib_ref] Algal toxicity of antibacterial agents used in intensive farming, Halling-Sørensen [/bib_ref] [bib_ref] Pharmaceutical antibiotic compounds in soils -A review, Thiele-Bruhn [/bib_ref]. Interestingly, functional metagenomics have revealed that, as the major resistance source against β-lactams, β-lactamase encoding genes were abundant even in undisturbed soil absent of anthropogenic selective pressure, contributing to a massive reservoir for genetic exchange among soil microflora [bib_ref] Functional metagenomics reveals diverse beta-lactamases in a remote Alaskan soil, Allen [/bib_ref]. Given our examination of fungal hydrolytic lactamases, we propose an ecological model [fig_ref] FIGURE 1 |: Basic lactam structures with differing ring sizes [/fig_ref] centering on the production and function of both lactams and lactamases produced by plants, bacteria, and fungi. We expand the conventional focus beyond that of solely bacterial β-lactamases and instead propose a more generic ecological model linking lactam production with hydrolytic functions of organismal lactamases. Lactam antibiotics presumably benefit their producers by securing ecological niches, whereas numerous lactam producers have also developed hydrolytic lactamases postulated to combat antibiosis. For example, soil-associated fungi typically possess more lactamase encoding genes than those from environments with lower microbial diversity since soil environments contain significant antibiotic diversity. Analysis of Fusarium species provides the foundation for our hypothesis that soil fungi frequently utilize lactamases in detoxification of xenobiotics, especially given Fusarium species' wide soil distribution, lactamase-rich genomes, and recent functional characterization of lactamase encoding genes. The general abundance and persistence of lactamase genes in fungal genomes suggests a significant role for these enzymes in the soil environment, presumably in protection from many as yet unknown xenobiotics. We have generated a large set of lactamase mutants in F. verticillioides and are conducting transcriptional and phenotypic analyses upon exposure to various lactam compounds in order to more thoroughly evaluate the role and activity of these lactamases, thus broadening our appreciation of both the lactam compounds and corresponding lactamases in terms of their diversity and impact on both bacterial and fungal communities. This work also has the potential to broaden our appreciation of environmental sources of antimicrobial resistance to include both bacteria and fungi, especially with regard to use of antibiotics in agriculture. # Author contributions All authors listed have made a substantial, direct and intellectual contribution to the work, and approved it for publication. # Funding This work was supported by USDA-ARS project 6040-42000-043-00D. Funding was provided to MG through the Department of Plant Pathology at the University of Georgia. [fig] FIGURE 1 |: Basic lactam structures with differing ring sizes. [/fig] [fig] FIGURE 2 |: Examples of lactam-containing fungal compounds. Lactam bonds are highlighted in red. [/fig] [fig] FIGURE 3 |: Fungicidal or fungistatic lactams. (1) N-thiolated β-lactams, artificial compounds that possess antifungal activity against Candida and other fungi by exerting powerful cytostatic effects that disrupt the structural integrity of cytoplasmic membranes (O'Driscoll et al., 2008); (2) Vince lactam, a versatile artificial chemical intermediate used in organic and medicinal chemistry that shows fungistatic effects against Botrytis cinerea, Penicillium citrium, and Aspergillus glaucus (Walczak et al., 2014); [/fig] [fig] FIGURE 6 |: Lactamase genes are abundant within some fungi. The number of annotated ORFs possessing a lactamase domain is shown for the top 40 fungi among all annotated fungal species identified from the NCBI Protein Database. Species are ranked by the abundance of lactamases. Fungi known to be soil-borne are displayed with red bars, those not clearly cited in literature as soil-borne fungi are in blue, and yeast along with selected obligate plant pathogens are in gray. Numbers of Fusarium lactamases are highlighted in yellow. [/fig] [fig] FIGURE 7 |: Orthological and syntenic analysis of Fusarium verticillioides, Fusarium graminearum, and F. oxysporum lactamase genes. Each column lists predicted β-lactamase orthologs in the three Fusarium species. Those sharing synteny of the adjacent 20 kb regions are shaded (10 kb upstream and 10 kb downstream). [/fig] [fig] FIGURE 11 |: Example of metallo-β-lactamase tertiary structures. (A) Predicted protein structure of FVEG_08291 exemplifying a general tertiary structure of metallo-β-lactamases. Motifs are color-coded according to relative conservation as shown in the scale bar. (B) Protein superimposition of FVEG_08291 and the 2R2D chain C from the Protein Data Bank (http://www.ebi.ac.uk/pdbe/), where cyan represents FVEG_08291 and magenta represents the 2R2D chain C. Front (C) and rear (D) view of proposed interactions between zinc ions and conserved MBL amino acids in FVEG_08291. [/fig] [table] TABLE 1 |: Classification of bacterial β-lactamases. [/table] [table] TABLE 2 |: Saccharomyces cerevisiae lactamase orthologs in three Fusarium species. Saccharomyces cerevisiae has seven lactamase genes, two of which (TRZ1 and YSH1) are essential. * * N/I represents no orthologs identified in these three Fusarium species. Fv, F. verticillioides; Fo, F. oxysporum; Fg, F. graminearum. [/table] [table] TABLE 3 |: β-lactamase domain-containing genes in three Fusarium genomes. [/table]

Esophageal Inlet Patch ## Clinical presentation A 65-year-old male was referred to radiology for a barium meal study with complaints of high dysphagia for solids with occasional choking and regurgitation. Chest radiography and electrocardiogram examinations were normal. The barium study was performed with rapid sequence filming of the pharynx during swallowing at 6 frames per second in anteroposterior (AP) and lateral projections. Because of the characteristic radiological findings, endoscopy was arranged the following day to confirm the diagnosis. ## Diagnosis Esophageal inlet patch (also called cervical inlet patch, ectopic or heterotopic gastric mucosa of the upper esophagus). ## Radiologic findings The barium swallow in the AP view taken at full cervical distension demonstrated two indentations in the barium column on the right [fig_ref] Figure 1: AP view of barium swallow showing two indentations [/fig_ref] , arrows) above the thoracic inlet. In between these indentations the barium column is bulging slightly outwards. These findings are characteristic of an esophageal inlet patch [bib_ref] Ectopic gastric mucosa in the upper esophagus: prevalence and radiologic findings, Takeji [/bib_ref] [bib_ref] Ectopic gastric mucosa in the upper esophagus: detection and radiographic findings, Ueno [/bib_ref]. On the lateral view, there is slight narrowing of the barium column at the thoracic inlet [fig_ref] Figure 2: Lateral view of barium swallow showing a mild narrowing of the esophageal... [/fig_ref] , arrow). The narrowing represents an esophageal stricture that is likely secondary to acid secretion by the inlet patch and is contributing to the patient's dysphagia. # Discussion Ectopic gastric mucosa can occur anywhere along the gastrointestinal (GI) tract. When it occurs in the upper esophagus, it is called "inlet patch" because of its location at or just distal to the upper esophageal sphincter. The inlet patch is considered a congenital anomaly found in 10% of the population with careful searching at endoscopybut it is often overlooked by endoscopists and radiologists and studies frequently report a prevalence between 0.1 and 3% [bib_ref] Ectopic gastric mucosa in the upper esophagus: prevalence and radiologic findings, Takeji [/bib_ref] [bib_ref] Heterotopic gastric mucosa in the cervical esophagus (inlet patch): endoscopic prevalence, histological..., Akbayir [/bib_ref] [bib_ref] Prevalence and clinical features of heterotopic gastric mucosa in the upper oesophagus, Maconi [/bib_ref] [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref]. Inlet patches are believed to be due to incomplete transformation from columnar to squamous 2 Radiology Research and Practice epithelium during embryonic development [bib_ref] Prevalence and clinical features of heterotopic gastric mucosa in the upper oesophagus, Maconi [/bib_ref]. Squamous transformation starts in the mid-esophagus and extends bidirectionally and incomplete terminal transformation at the proximal esophagus accounts for the postcricoid location of inlet patches [bib_ref] Inlet Patch: prevalence, histologic type, and association with esophagitis, Barrett esophagus, and..., Tang [/bib_ref]. Most inlet patches are solitary and extend longitudinally, affecting only part of the circumference, but some are annular and multiple lesions are not uncommon [bib_ref] Ectopic gastric mucosa in the upper esophagus: prevalence and radiologic findings, Takeji [/bib_ref] [bib_ref] Prevalence and clinical features of heterotopic gastric mucosa in the upper oesophagus, Maconi [/bib_ref]. Most inlet patches are largely asymptomatic, but in problematic cases complications related to acid secretion such as esophagitis, ulcer, web, and stricture may produce symptoms such as chest and throat pain, dysphagia, globus sensation, and shortness of breath [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref] [bib_ref] Inlet Patch: prevalence, histologic type, and association with esophagitis, Barrett esophagus, and..., Tang [/bib_ref] [bib_ref] Heterotopic gastric mucosa (inlet patch) in a patient with laryngopharyngeal reflux (LPR)..., Basseri [/bib_ref]. The size of the patch is related with dysphagia severity, possibly as a function of increased acid secretion [bib_ref] Heterotropic gastric mucosa (inlet patch): endoscopic prevalence, histopathological, demographical and clinical characteristics, Poyrazoglu [/bib_ref]. In some cases of inlet patch ulcer, serious and life-threatening sequelae such as hemorrhage, perforation, and tracheoesophageal fistula may occur [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref]. Amongst those with concurrent inlet patch and gastric H. pylori, 73% will have an infected inlet patch [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref] which may exacerbate complications and related symptoms. Chronic cough and hoarseness have been reported in association with inlet patches, presumably due to acid irritation of the airways and vocal cords [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref] [bib_ref] Inlet patch of gastric mucosa in upper esophagus causing chronic cough and..., Silvers [/bib_ref]. Adenocarcinoma may arise in the ectopic gastric mucosa but this is rare and is considered sporadic. In contrast to Barrett's esophagus there is no increased risk for adenocarcinoma associated with inlet patches as they are not metaplastic [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref]. The diagnosis of inlet patch is strongly suggested by characteristic findings on barium swallow [bib_ref] Ectopic gastric mucosa in the upper esophagus: prevalence and radiologic findings, Takeji [/bib_ref] [bib_ref] Ectopic gastric mucosa in the upper esophagus: detection and radiographic findings, Ueno [/bib_ref]. The lesions are most evident when the cervical esophagus is at maximum distension following the opening of the upper esophageal sphincter. Characteristic findings are discussed in [bib_ref] Ectopic gastric mucosa in the upper esophagus: prevalence and radiologic findings, Takeji [/bib_ref] [bib_ref] Ectopic gastric mucosa in the upper esophagus: detection and radiographic findings, Ueno [/bib_ref]. The most common pattern consists of two small indentations on the wall of the esophagus. Alternatively the indentations may be more prominent with an intervening bulge away from the esophageal lumen, as was noted in the images of the case presented here or there may be only a single indentation. Other possible findings reflect the prominent border of the inlet patch and include rim-like shadows and irregular outlines. The diagnosis of inlet patch is confirmed via endoscopy with biopsy. The lesion will be seen more often by endoscopists whose custom is to withdraw the scope very slowly through the upper sphincter in order to inspect the arytenoids and vocal cords. At endoscopy, the lesion appears salmon-coloured and velvety and is easily distinguished from the normal grey-white squamous epithelium of the esophagus [fig_ref] Figure 3: Endoscopic appearance of an annular inlet patch from a different patient [/fig_ref] [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref]. Inlet patches range from 0.2 to 5 cm and can be round or oval with a flat, slightly raised, or depressed surface and may have heaped margins most often on the lateral or posterior surfaces [bib_ref] Heterotopic gastric mucosa in the upper esophagus: a prospective study of 33..., Jacobs [/bib_ref]. The prominent margins correlate with the radiological findings of indentations and rim-like shadows on barium swallow. Histopathology provides the definitive diagnosis by demonstrating gastric mucosa adjacent to normal esophageal mucosa [fig_ref] Figure 4: Histology of a portion of an inlet patch from a different patient... [/fig_ref]. Histopathological studies have demonstrated that oxyntic mucosa (gastric body-like with acid-secreting parietal cells) is the most common histologic subtype of esophageal ectopic gastric mucosa but cardiac, antral, and mixed types also occur [bib_ref] Heterotopic gastric mucosa in the cervical esophagus (inlet patch): endoscopic prevalence, histological..., Akbayir [/bib_ref] [bib_ref] Inlet Patch: prevalence, histologic type, and association with esophagitis, Barrett esophagus, and..., Tang [/bib_ref]. A treatment strategy based on symptoms and underlying pathology is outlined in [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref]. There is no treatment required for asymptomatic inlet patches. Affected individuals who are symptomatic may find relief with the use of proton pump inhibitors. Strictures and webs are treated with serial dilatation [bib_ref] Heterotopic gastric mucosa in the cervical esophagus (inlet patch): endoscopic prevalence, histological..., Akbayir [/bib_ref] [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref] but should include biopsy to rule out malignancy [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref]. Ablation of inlet patches has been shown to relieve globus [bib_ref] Inlet patch of gastric mucosa in upper esophagus causing chronic cough and..., Silvers [/bib_ref] and has been used to successfully treat inlet patch dysplasia although its routine use in this context has not been determined [bib_ref] Heterotopic gastric mucosa of the esophagus: literature-review and proposal of a clinicopathologic..., Von Rahden [/bib_ref]. As there was no evidence of mechanical obstruction, the cause of our patient's symptoms were thought to be secondary to esophageal irritation from acid-secretion. He responded well to treatment with a proton pump inhibitor. [fig] Figure 1: AP view of barium swallow showing two indentations (arrows) above the thoracic inlet and a slight lateral bulge of the esophageal lumen between the indentations. [/fig] [fig] Figure 2: Lateral view of barium swallow showing a mild narrowing of the esophageal lumen (arrow) at the level of the thoracic inlet. [/fig] [fig] Figure 3: Endoscopic appearance of an annular inlet patch from a different patient. The ectopic gastric mucosa is salmon-coloured and velvety and is easily distinguished from the normal grey-white esophageal epithelium. The raised border of the lesion corresponds to the radiological findings of esophageal indentations. Image reprinted with permission from Medscape.com, 2009. Available at: http://www.medscape.com/viewarticle/405480. [/fig] [fig] Figure 4: Histology of a portion of an inlet patch from a different patient showing gastric mucosa adjacent to normal esophageal mucosa with stratified squamous epithelium (top left). The gastric mucosa shown here is antral-type as there are no oxyntic (acidsecreting parietal cell) glands. [/fig]

The effect of the VKORC1 promoter variant on warfarin responsiveness in the Saudi WArfarin Pharmacogenetic (SWAP) cohort Warfarin is a frequently prescribed oral anticoagulant with a narrow therapeutic index, requiring careful dosing and monitoring. However, patients respond with significant inter-individual variability in terms of the dose and responsiveness of warfarin, attributed to genetic polymorphisms within the genes responsible for the pharmacokinetics and pharmacodynamics of warfarin. Extensive warfarin pharmacogenetic studies have been conducted, including studies resulting in genotype-guided dosing guidelines, but few large scale studies have been conducted with the Saudi population. In this study, we report the study design and baseline characteristics of the Saudi WArfarin Pharmacogenomics (SWAP) cohort, as well as the association of the VKORC1 promoter variants with the warfarin dose and the time to a stable INR. In the 936 Saudi patients recruited in the SWAP study, the minor allele C of rs9923231 was significantly associated with a 8.45 mg higher weekly warfarin dose (p value = 4.0 × 10 -46 ), as well as with a significant delay in achieving a stable INR level. The addition of the rs9923231 status to the model, containing all the significant clinical variables, doubled the warfarin dose explained variance to 31%. The SWAP cohort represents a valuable resource for future research with the objective of identifying rare and prevalent genetic variants, which can be incorporated in personalized anticoagulation therapy for the Saudi population.Warfarin is a frequently prescribed oral anticoagulant agent inhibiting the synthesis of the Vitamin K dependent clotting factors II, VII, IX, X as well as anticoagulation factors protein C and S. It also causes anticoagulation by inhibiting the Vitamin K-epoxide reductase complex, which result in the accumulation of reduced vitamin K2, 3-epoxide and vitamin K hydroquinone 1 . Warfarin is indicated for the treatment or prophylaxis of patients with various disease states, including venous thromboembolic events 2 , atrial fibrillation and flutter 3 , cardiac surgery for valve replacement and orthopedic surgery 4,5 . Warfarin exhibits a narrow therapeutic index and is monitored through the International Normalized Ratio (INR), which measures the ability of the blood to clot. The optimal effect of warfarin, for most of the indications, is when the INR ranges from 2 to 3, except for metallic atrial valve replacement, in which a higher INR value is required 6 .Several studies reported ethnic differences in the warfarin response and dosage requirements 7-9 . Even for populations with very close ancestry within the Middle East and North Africa (MENA) region, differences in open 1 www.nature.com/scientificreports/ allelic frequencies and their impact on warfarin dosing has been documented [bib_ref] The impact of genetic and non-genetic factors on warfarin dose prediction in..., Bader [/bib_ref]. This wide inter-individual variability in the warfarin dose is attributed to several factors, including allelic differences of genetic variants in the warfarin metabolizing enzyme, and environmental factors within each population [bib_ref] Genetic determinants of response to warfarin during initial anticoagulation, Schwarz [/bib_ref] [bib_ref] Contribution of age, body size, and CYP2C9 genotype to anticoagulant response to..., Kamali [/bib_ref]. Genetic variants within cytochrome P450 2C9 (CYP2C9) and the vitamin K-epoxide reductase complex (VKORC1) enzyme explain approximately 50% of the dose variability [bib_ref] Clinical pharmacogenetics implementation consortium guidelines for CYP2C9 and VKORC1 genotypes and warfarin..., Johnson [/bib_ref]. Most of the pharmacogenetic guidelines consider variants within two genes, CYP2C9*2, CYP2C9*3, and VKORC1:c.−1639C>T (rs9923231) [bib_ref] Clinical Pharmacogenetics Implementation Consortium (CPIC) guideline for pharmacogenetics-guided warfarin dosing: 2017 update, Johnson [/bib_ref]. The differences in the percentage of variance in the warfarin dose explained by VKORC1 in a population are, to a large degree, due to rs9923231 [bib_ref] Warfarin pharmacogenetics: A single VKORC1 polymorphism is predictive of dose across 3..., Limdi [/bib_ref]. This variant is significantly associated with the warfarin dose in several populations, including Indians, Chinese, Brazilian, Turkish, Russian, and Emiratis [bib_ref] Warfarin dose model for the prediction of stable maintenance dose in Indian..., Gaikwad [/bib_ref] [bib_ref] Warfarin dosage response related pharmacogenetics in chinese population, Li [/bib_ref] [bib_ref] Algorithm for predicting low maintenance doses of warfarin using age and polymorphisms..., De Oliveira Magalhães Mourão [/bib_ref] [bib_ref] VKORC1 and CYP2C9 polymorphisms are associated with warfarin dose requirements in Turkish..., Oner Ozgon [/bib_ref] [bib_ref] CYP2C9 and VKORC1 genotyping for the quality of long-standing warfarin treatment in..., Panchenko [/bib_ref] [bib_ref] Vkorc1 variants as significant predictors of Warfarin dose in Emiratis, Al-Mahayri [/bib_ref]. All the studies indicated a significant influence on the warfarin response, accounting for 11-32% of the variability in the warfarin dose [bib_ref] Warfarin pharmacogenetics: A single VKORC1 polymorphism is predictive of dose across 3..., Limdi [/bib_ref] [bib_ref] Warfarin dose model for the prediction of stable maintenance dose in Indian..., Gaikwad [/bib_ref] [bib_ref] Warfarin dosage response related pharmacogenetics in chinese population, Li [/bib_ref] [bib_ref] Algorithm for predicting low maintenance doses of warfarin using age and polymorphisms..., De Oliveira Magalhães Mourão [/bib_ref] [bib_ref] VKORC1 and CYP2C9 polymorphisms are associated with warfarin dose requirements in Turkish..., Oner Ozgon [/bib_ref] [bib_ref] CYP2C9 and VKORC1 genotyping for the quality of long-standing warfarin treatment in..., Panchenko [/bib_ref] [bib_ref] Vkorc1 variants as significant predictors of Warfarin dose in Emiratis, Al-Mahayri [/bib_ref]. However, few studies with the MENA populations, and particularly the Saudi population, have been conducted to investigate the pharmacogenetic interaction of warfarin responsiveness. Establishing a large cohort of warfarin users would provide opportunities to identify novel risk alleles for drug responses and prevalent diseases, especially with the high consanguinity rate within the Saudi population [bib_ref] Genetics and genomic medicine in Saudi Arabia, Alkuraya [/bib_ref]. Thus, the aim of this study was to report the cohort profile of the Saudi WArfarin Pharmacogenomics (SWAP), as a large prospective cohort for warfarin pharmacogenomic studies, and to assess the value of this cohort by reporting the association of rs9923231 to warfarin effectiveness. Riyadh and Jeddah, Saudi Arabia. All patients were Saudi nationals, older than 18 years, and for whom warfarin therapy was initiated or patients using warfarin with an INR 2-3. Patients were excluded if their baseline anticoagulation profile was prolonged (INR more than 1.5), aPTT > 1.5-2 times the normal value, baseline bilirubin more than 2.4 gm/dl, had a mechanical heart valve replacement that requires the INR target to be above 3, receiving chemotherapy, and had a confirmed diagnosis of HIV or hepatitis A, B, C. An EDTA peripheral blood sample was drawn for genetic analysis and the clinical variables were retrieved from the hospital's electronic records system during the hospital admission. The data included demographic and anthropometric variables such as age, gender, weight, and height. The clinical data included comorbidities, renal, liver, and thyroid functions, drug interactions, hemoglobin levels and the anticoagulation profile. In this study, patients were followedup for 10 days only, regardless of whether the participant was admitted or an outpatient. All the variables were compiled in an electronic database for subsequent analysis. # Methods DNA extraction and genotyping assay. All patients signed a written informed consent before peripheral blood samples were collected. Genomic DNA was extracted from the peripheral blood using a Qiagen DNeasy kit (Qiagen, Germany) (spin-column protocol) following the manufacturer's protocol. In brief, the whole blood sample was lysed using a lysis buffer, the DNA was bonded through a silica binding column and eluted through a similar column using an elution buffer. DNA purity and quantity was assessed using the Nanodrop™ spectrophotometer 8,000 and BR Qubit 3 fluorometer respectively, high quality DNA samples are used for genotyping of rs9923231 (VKOR1C −1639 C>T) using a ready-made fluorescence probe assay (C_30403261_20) by Thermo Fisher Scientific, MA, USA. All the genotyping reactions were done with the TaqMan genotyping master mix (Lot No. 0074819). The genotyping procedure was performed following the standard protocol. In brief, a total volume of 25 µl was added in each well of the 96-well plates, as well as 1.25 µl of genotyping master mix, 1.25 µl of genotyping assay, 9.25 µl of nuclease-free water and 2 µl of 20 ng/µl of DNA. The amplification protocol started with a pre-read stage at 60 °C/30 s followed by a hold stage at 95 °C/10 min; the PCR stage is 95 °C/15 s followed by 60 °C/1 min for 50 cycles. The final stage is a post-read stage at 60 °C/30 s. Reporter dyes for qPCR were detected and analyzed by a real-time qPCR ABI QuantStodio 6 FLEX system with TaqMan Genotyper Software. Statistical analysis. Continuous variables are summarized as median with interquartile ranges (IQR), and categorical variables as total number and percentage. The allelic frequency of the rs9923231 summary was done in PLINK to calculate the minor allele frequency (MAF) and Hardy-Weinberg Equilibrium p value [bib_ref] PLINK: A tool set for whole-genome association and population-based linkage analyses, Purcell [/bib_ref]. A stepwise regression model was used to select significant non-genetic factors by choosing a p value threshold of 0.25 for entry and 0.1 for exiting the model. Only factors with a significant p value < 0.05 were kept in the non-genetic model. The genetic model included the same variables, plus the rs9923231 genotype as coded by 0, 1, and 2 for the genotypes TT, CT, and CC, respectively. The R 2 and adjusted R 2 were calculated for the two models. A linear regression model was used to test for the association of rs9923231 with the weekly average dose after adjusting for age, gender, body mass index and smoking status. The average daily dose was calculated as the average warfarin dose from day 3 to day 10. The Cox regression Hazard model has been adopted by several studies to evaluate the effect of genotypes on the time required to reach a stable INR [bib_ref] Prospective evaluation of a pharmacogenetics-guided warfarin loading and maintenance dose regimen for..., Gong [/bib_ref] [bib_ref] Effect of genotype-guided warfarin dosing on clinical events and anticoagulation control among..., Gage [/bib_ref]. In this study, a Cox regression model was performed to determine the hazard ratio (HR) for achieving the target INR, and a Kaplan-Meier Survival curve was plotted for the different genotypes and log-rank statistics were computed to test the homogeneity of the estimated survival function for the groups [bib_ref] Effect of genotype-guided warfarin dosing on clinical events and anticoagulation control among..., Gage [/bib_ref]. Scientific RepoRtS | (2020) 10:11613 | https://doi.org/10.1038/s41598-020-68519-9 www.nature.com/scientificreports/ # Results Baseline characteristics. During the study recruitment period, a total of 2,581 patients were screened as potential participants in the study [fig_ref] Figure 1: Flow chart of the participants screening step [/fig_ref]. Of this group, 936 patients fulfilled the inclusion criteria and included in the study. The sample was classified according to the warfarin status on the day of recruitment, to either naïve patients (n = 602), who have never been prescribed warfarin before, or to ex-user (n = 334), who were already on warfarin treatment on the day of recruitment. The baseline characteristics of the sample are displayed in Tables 1 and 2. The average age was 62.4 ± 18.9 years, and 55.2% were female. The most prevalent indications for warfarin were atrial fibrillation (AF, 44.7%), pulmonary embolism (PE, 18.7%), and deep vein thrombosis (DVT, 18.3%). The most frequent comorbidities were hypertension (75%), diabetes (54%), and hyperlipidemia (42%). Genotyping rs9923231. The minor allele frequency of rs9923231 allele C was 0.43, and the genotypic frequencies were 0.21, 0.45, and 0.34 for genotypes CC, CT, and TT, respectively [fig_ref] Table 3: Genotypic frequency of rs9923231 and association with average weekly dose of warfarin [/fig_ref]. The genotypic frequency was not significantly different between the groups on initiation or in the maintenance phase (p value = 0.27). The average weekly dose of warfarin was significantly different between the three genotypes (p value = 1.81E-40), with 35.28 mg (95% CI 33.48-37.08), 25.45 mg (95% CI 24.23-26.66), and 18.73 mg (95% CI 17.33-20.14) for the groups of CC, CT, and TT carriers, respectively [fig_ref] Figure 1: Flow chart of the participants screening step [/fig_ref]. In addition, the rs9923231 genotype was significantly associated with the daily warfarin dose; each copy of the C allele was associated with a 1.16 mg higher dose of warfarin (p value = 3.55 × 10 -45 , [fig_ref] Table 3: Genotypic frequency of rs9923231 and association with average weekly dose of warfarin [/fig_ref] after adjusting for age, BMI, gender, and smoking status. At a week interval, the warfarin dose was higher by an average of 8.45 mg/week (p value = 4.0 × 10 -46 ) for each additional allele-C copy [fig_ref] Figure 2: Average of warfarin weekly dose by rs29923231 genotype status [/fig_ref]. Time to stable INR. The Cox regression model indicated a significant association of the rs9923231 genotype with the time to a stable INR [fig_ref] Figure 2: Average of warfarin weekly dose by rs29923231 genotype status [/fig_ref]. Patients with the CC genotype had a statistically significant delay Warfarin dose. In the subgroup of 602 warfarin naïve patients, the C allele of rs9923231 was significantly associated with an average of 1.14 mg higher dose of warfarin (p value = 6.21E−31, [fig_ref] Table 3: Genotypic frequency of rs9923231 and association with average weekly dose of warfarin [/fig_ref]. At day 10, 61% of the CC carriers achieved a stable INR compared to 78% and 82% of the CT and TT carriers, respectively . The categorical variables of the baseline characteristics of the patients. Bold shows a statistical significant differences between naïve and ex-user group. www.nature.com/scientificreports/ compared to CT, and by 1.76 (95% confidence interval 1.35-2.32, p value = 0.00005) compared to TT, respectively. In the subgroup of 334 maintenance dose patients, the rs9923231-C was also significantly associated with an average of 1.24 mg higher dose of warfarin (p value = 9.37E−16, [fig_ref] Table 3: Genotypic frequency of rs9923231 and association with average weekly dose of warfarin [/fig_ref]. However, there were no statistically significant difference between the three genotypes in maintaining a stable INR during the first 10 days of observation (p value = 0.75, [fig_ref] Figure 4: Number of days to reach a stable INR for [/fig_ref]. Non-genetic factors. All non-genetic factors were entered in the stepwise multiple regression model to identify the clinical variables significantly associated with the warfarin dose. The stepwise regression identified seven factors namely, age, weight, status of warfarin, stroke as an indication, the laboratory measurements of red blood cells (RBC), Gamma-Glutamyl Transferase (GTP), and Alkaline phosphatase [fig_ref] Table 4: Stepwise parameters for the impact of non-genetic and genetic models on the... [/fig_ref]. These factors explained 14% of the daily warfarin dose. The addition of rs9923231 into the model explained an additional 17%, with total of 31% variance explained by the full genetic model [fig_ref] Table 4: Stepwise parameters for the impact of non-genetic and genetic models on the... [/fig_ref]. www.nature.com/scientificreports/ # Discussion The SWAP cohort is the largest cohort of Saudi warfarin using patients with the aim of studying the pharmacogenetics of warfarin responsiveness. In this relatively large cohort, we found a statistically significant association between rs9923231 and the average daily warfarin dose as well as the number of days to achieve a stable INR. The addition of the rs9923231 genotype to the warfarin dose prediction model, that included non-genetic factors, doubled the explained variation to 31%. The rs9923231 was a statistically significant factor that determined the number of days required to achieve a stable INR when initiating warfarin, but this significance was not found with the group receiving a maintenance dose. www.nature.com/scientificreports/ The role of genetic variants within VKORC1 is known and have been incorporated in genotype-based dosing algorithms in several populations [bib_ref] Clinical pharmacogenetics implementation consortium guidelines for CYP2C9 and VKORC1 genotypes and warfarin..., Johnson [/bib_ref]. Essentially, warfarin antagonizes the Vitamin K-dependent clotting pathway, in which the VKORC1 gene product, VKORC1 protein, is the rate-limiting step in Vitamin K recycling [bib_ref] Increased production of functional recombinant human clotting factor IX by baby hamster..., Wajih [/bib_ref]. As the rs9923231 is located in the promoter region of VKORC1, it alters the promoter activity and the transcription factor binding site, leading to a reduction by almost 44% in the luciferase activity of the T allele compared to the C allele [bib_ref] A novel functional VKORC1 promoter polymorphism is associated with inter-individual and inter-ethnic..., Yuan [/bib_ref]. This polymorphism is the most prevalent polymorphism in VKORC1 associated with the warfarin dose and sensitivity [bib_ref] Clinical pharmacogenetics implementation consortium guidelines for CYP2C9 and VKORC1 genotypes and warfarin..., Johnson [/bib_ref] [bib_ref] Effect of VKORC1 haplotypes on transcriptional regulation and warfarin dose, Rieder [/bib_ref] [bib_ref] Mutations in VKORC1 cause warfarin resistance and multiple coagulation factor deficiency type..., Rost [/bib_ref]. The International Warfarin Pharmacogenetics Consortium (IWPC) recommend reducing the weekly warfarin dose for carriers of the C allele. Limited studies investigated the impact of the pharmacogenetic effect on warfarin treatment in Saudi patients. A study with 112 Saudi patients reported an allele frequency estimate of 45% for allele C, similar to the current study [bib_ref] Genetic risk assessment towards warfarin application: Saudi Arabia study with a potential..., Al-Saikhan [/bib_ref]. Other studies were conducted with healthy volunteers to estimate the allelic frequency [bib_ref] A European spectrum of pharmacogenomic biomarkers: Implications for clinical pharmacogenomics, Mizzi [/bib_ref] [bib_ref] Genotyping of CYP2C9 and VKORC1 in the arabic population of Al-Ahsa, Saudi..., Alzahrani [/bib_ref]. Comparing 499 healthy Saudi participants to 1,105 Europeans, and 106 South Africans, indicated the allele frequencies for the C allele as 0.46, 0.42, and 0.36, respectively [bib_ref] A European spectrum of pharmacogenomic biomarkers: Implications for clinical pharmacogenomics, Mizzi [/bib_ref]. Based on the allele frequencies, this study predicted a statistically significant difference in the warfarin dose between the three populations, with the Saudi population having an average warfarin dose equal to [bib_ref] A randomized trial of pharmacogenetic warfarin dosing in naïve patients with non-valvular..., Pengo [/bib_ref] The IWPC dosing guideline recommends decreasing the average dose of warfarin for patients with the rs9923231 TT and CT genotypes by − 16.14 mg and − 8.97 mg/week, respectively, compared to patients with similar characteristics but a carrier of the CC genotype. In our study, the average of warfarin dose for TT and CT carriers was lowered by − 16.54 and − 9.83 mg/week, respectively. Although these estimates are based solely on the VKORC1 status with no consideration of other important factors such as the CYP2C9 genotype, it indicates that patients treated by the traditional dosing method, have converged to groups with an average dose close to the IWPC dosing guidelines. This does not imply that the full dosing algorithm can be applied in the Saudi population, as other factors could be different between the populations. Other rare variants within VKORC1 and other important pharmacogenes (VIP) could play major roles in populations differences [bib_ref] Influence of common and rare genetic variation on warfarin dose among African..., Liu [/bib_ref]. It must be considered that the clinical value of a genotype-based dosing algorithm, over the standard dosing approach, is still unclear [bib_ref] Effect of genotype-guided warfarin dosing on clinical events and anticoagulation control among..., Gage [/bib_ref] [bib_ref] A randomized trial of genotype-guided dosing of warfarin, Pirmohamed [/bib_ref] [bib_ref] A randomized trial of pharmacogenetic warfarin dosing in naïve patients with non-valvular..., Pengo [/bib_ref]. To determine whether this result will translate into significant clinical benefits or whether the work describes interesting rare genetic variants remains to be examined. In this study, we achieved a relatively large sample size of warfarin patients from an under-represented population. The SWAP cohort provides a valuable resource for future studies to develop genotype-based dosing algorithms, targeting the Saudi population. Although we only tested the pharmacogenetic impact of VKORC1 on the warfarin response, the study serves as proof of concept to justify future research to assess the full spectrum of genetic variants using advanced technologies, such as Next-generation Sequencing. One limitation of this study is the fact that we followed-up the patients for 10 days only. During which, it is difficult to draw an informative interpretation about the quality of clinical setting in patients, taking into consideration that the majority of our patients have never been exposed to warfarin at the time of enrollment in the study. Nevertheless, longer followup analysis will be conducted in future studies by following the patient electronic health record. In conclusion, SWAP represents an unprecedented resource as the largest national cohort of warfarin using patients to support a range of studies, with the ultimate goal of identifying rare and prevalent variants to develop personalized anticoagulation treatment. We reported the association of the VKORC1 promoter variant with the warfarin dose and time to a stable INR in the Saudi population. The findings establish a baseline for additional studies to assess the impact of genotype-guided warfarin dosing by using the SWAP materials. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creat iveco mmons .org/licen ses/by/4.0/. [fig] Figure 1: Flow chart of the participants screening step. [/fig] [fig] Figure 2: Average of warfarin weekly dose by rs29923231 genotype status. [/fig] [fig] Figure 3: Time to stable INR between the three genotypes. Scientific RepoRtS | (2020) 10:11613 | https://doi.org/10.1038/s41598-020-68519-9 [/fig] [fig] Figure 4: Number of days to reach a stable INR for (A) Warfarin Naïve group and (B) Warfarin ex-user patients. [/fig] [table] Table 3: Genotypic frequency of rs9923231 and association with average weekly dose of warfarin. *Model is adjusted for age, gender, BMI, and smoking status. **Chi Square test for the difference in genotypic frequencies between the initiation and maintenance groups is not significant (p value = 0.273). Bold shows a statistical significant differences between naïve and ex-user group. [/table] [table] Table 4: Stepwise parameters for the impact of non-genetic and genetic models on the warfarin daily dose. [/table]

Spotlight on adalimumab in the treatment of active moderate-to-severe hidradenitis suppurativa Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory skin disease that affects the hair follicles of the aprocrine gland-bearing anatomical areas of the body. It is characterized by deep painful nodules and abscesses that rupture and contribute to the formation of sinus tracks and scarring. The management of HS is based on the assessment of disease severity and a combination of medical and surgical treatment according to the European Guidelines. Adalimumab, a recombinant, fully humanized, anti-tumor necrosis factor alpha (anti-TNF-α) monoclonal antibody, is the only officially approved treatment for the management of moderateto-severe HS. Case reports, concerning 42 patients who received adalimumab for severe HS (with the standard dose regimen for psoriasis), reported a cumulative response rate of 58% (≥50% in 23 patients) with a relapse rate of 71% (10 out of 14 patients). The most recent and most well-powered phase III, randomized placebo-controlled trials for the evaluation of the efficacy and safety of adalimumab in treatment of moderate-to-severe HS (PIONEER studies I and II) showed that the Hidradenitis Suppurativa Clinical Response (HiSCR) rate at week 12 was significantly higher for patients randomized to adalimumab compared to placebo. Adverse events were comparable to placebo. In conclusion, adalimumab, to date, holds the most robust data regarding treatment efficacy in HS. Larger, registry-based studies are needed to further establish the efficacy and safety profile of this anti-TNF-α agent in HS. # Introduction Hidradenitis suppurativa (HS) is a chronic, recurrent, inflammatory skin disease that affects the hair follicles of the aprocrine gland-bearing anatomical areas of the body. [bib_ref] Comordidities of hidradenitis suppurativa (acne inversa), Fimmel [/bib_ref] It is characterized by deep painful nodules and abscesses that rupture and contribute to the formation of sinus tracks and scarring. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] The axillae, inguinal, anogenital regions and mammary, and inframammary areas are the most commonly affected parts of the body. [bib_ref] An update on hidradenitis suppurativa (part I): epidemiology, clinical aspects and definition..., Martorell [/bib_ref] The diagnosis of the disease is based on the following findings: history of recurrent, painful, suppurating lesions in intertrigenous body areas for two or more times in 6 months. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] The exact prevalence of the disease is uncertain, though, it is believed that it ranges between 1% and 4%. [bib_ref] Epidemiology and scope of the problem, Jemec [/bib_ref] Its estimated incidence -according to a study by Vazquez et al -reaches 6.0 cases per 100,000 person years. [bib_ref] Prevalence and factors associated with hidradenitis suppurativa: results from two case controls..., Revuz [/bib_ref] [bib_ref] The prevalence of hidradenitis suppurativa and its potential precursor lesions, Jemec [/bib_ref] [bib_ref] Incidence of hidradenitis suppurativa and associated factors: a population-based study of Olmsted..., Vazquez [/bib_ref] HS most frequently occurs in young adults during the second and third decade of their life. [bib_ref] Clinical charecterisitcs of a series of 302 French patients with hidradenitis suppurativa,..., Cannoui-Poitrine [/bib_ref] [bib_ref] Natural history, presentation and diagnosis of hidradenitis suppurativa, Michelletti [/bib_ref] There is a characteristic female predominance (female to male ratio 3:1), although males tend to suffer from more severe symptoms of the disease. 10 submit your manuscript | www.dovepress.com ## Dovepress ## Dovepress ## 368 Fotiadou et al Follicular occlusion is now considered the main pathogenetic event in HS. [bib_ref] Pathophysiology of hidradenitis suppurativa: an update, Prens [/bib_ref] Evidently, an imbalance of the immune system which precedes and/or follows follicular occlusion -in genetically predisposed individuals -also plays an important role in course of the disease. [bib_ref] An update on hidradenitis suppurativa (part I): epidemiology, clinical aspects and definition..., Martorell [/bib_ref] [bib_ref] Hidradenitis suppurativa a review with a focus on treatment data, Barlev [/bib_ref] [bib_ref] Hidradenitis suppurativa: the role of immune dysregulation, Kelly [/bib_ref] [bib_ref] Hidradenitis supurativa: the role of deficient cutaneous innate immunity, Dreno [/bib_ref] A genetic background, expressed with an autosomal dominant inheritance pattern, is present in about 40% of patients suffering from HS. [bib_ref] Gamma-secretase gene mutations in familial acne inversa, Wang [/bib_ref] Certain trigger factors such as smoking ( 70%-88.9% of cases), obesity (50% of cases), mechanical friction and to a lesser extent, hormones, drugs, and depilation seem to contribute to the manifestation of this entity. [bib_ref] Hidradenitis Suppurativa: a comprehensive review, Alikhan [/bib_ref] [bib_ref] Epidemiology of hidradenitis suppurativa: prevalence, pathogenesis, and factors associated with the development..., Deckers [/bib_ref] [bib_ref] Cigarette smoking as a triggering factor of hidradenitis suppurativa, Konig [/bib_ref] [bib_ref] Objective scoring of hidradenitis suppurativa reflecting the role of tobacco smoking and..., Sartorius [/bib_ref] [bib_ref] Hidradenitis suppurativa, Margesson [/bib_ref] A matter of controversy among authors is the possibility that the alteration of microbial flora and the formation of bacterial biofilms could act as a predisposing factor for HS patients. [bib_ref] Hidradenitis suppurativa: current views on epidemiology, pathogenesis and pathophysiology, Michelleti [/bib_ref] [bib_ref] Alterations in leucocyte subsets and histomorphology in normal-appearing perilesional skin and early..., Van Der Zee [/bib_ref] [bib_ref] Considering hidradenitis suppurativa as a bacterial biofilm disease, Kathju [/bib_ref] HS has been associated with several comorbid autoinflammatory disorders (inflammatory bowel disease and spondyloarthropathy) as well as pyoderma gangrenosum and epithelial tumors (squamous cell carcinomas). [bib_ref] Comordidities of hidradenitis suppurativa (acne inversa), Fimmel [/bib_ref] [bib_ref] Prevalence and factors associated with hidradenitis suppurativa: results from two case controls..., Revuz [/bib_ref] [bib_ref] Diseases associated with hidradenitis suppurativa: Part 2 of a series on hidradenitis, Scheinfield [/bib_ref] [bib_ref] Hidradenitis suppurativa and inflammatory bowel disease: are they associated? Results from a..., Van Der Zee [/bib_ref] [bib_ref] Hidradenitis suppurativa associated with spondyloarthritis-results from a multicenter national prospective study, Richette [/bib_ref] HS patients experience a negative impact on physical, social, and emotional aspects of their life. [bib_ref] The psychological impact of hidradenitis suppurativa, Gooderham [/bib_ref] As a consequence, patients have a poor quality of life that is assessed using the Dermatology Life Quality Index (DLQI), whose value ranges between 8.4 and 12.7 in several studies. [bib_ref] Morbidity in patients with hidradenitis supurativa, Von De Werth [/bib_ref] [bib_ref] Depression in patients with hidradenitis suppurativa, Onderdijk [/bib_ref] [bib_ref] Psycophysical aspects of hidradenitis suppurativa, Matusiak [/bib_ref] ## Advances in the management of hs The strategic management of HS is based on the assessment of disease severity and a combination of medical and surgical treatment according to the European Guidelines. 2,31 All patients with HS could benefit from adjuvant therapy which may include pain management, treatment of super-infections, as well as weight loss and smoking cessation. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] [bib_ref] Evidence-based approach to the treatment of hidradenitis suppurativa/acne inversa, based on the..., Gulliver W [/bib_ref] In terms of surgical intervention, procedures such as deroofing, laser, local excision, or even wide excision could be implemented depending on the type and extent of abscesses and the severity of scaring in each individual case. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] [bib_ref] Evidence-based approach to the treatment of hidradenitis suppurativa/acne inversa, based on the..., Gulliver W [/bib_ref] The management of the inflammatory component of the HS is based on medical treatment. For mild disease (1-2 small abscesses or inflammatory nodules), topical clindamycin 1% twice daily may be helpful. If the patient does not respond or has moderate-to-severe disease, the administration of tetracycline 500 mg po (orally) twice daily for 4 months or even the combination of clindamycin 300 mg po twice daily along with rifampicin 600 mg once daily for 10 weeks should be considered. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] [bib_ref] Evidence-based approach to the treatment of hidradenitis suppurativa/acne inversa, based on the..., Gulliver W [/bib_ref] The next treatment choice in the occasion of no improvement is adalimumab 160 mg at week 0, 80 mg at week 2, and then 40 mg subcutaneously (sc) every week. [bib_ref] Evidence-based approach to the treatment of hidradenitis suppurativa/acne inversa, based on the..., Gulliver W [/bib_ref] If this treatment is effective, it should be maintained as long as the lesions are present. [bib_ref] Evidence-based approach to the treatment of hidradenitis suppurativa/acne inversa, based on the..., Gulliver W [/bib_ref] In the case of no response, second-line (infliximab, acitretin) and third-line (cyclosporine, dapsone, hormones, isotretinoin, alitretinoin, colchicines) treatments should be tried. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] [bib_ref] Evidence-based approach to the treatment of hidradenitis suppurativa/acne inversa, based on the..., Gulliver W [/bib_ref] Rationale for the use of adalimumab in the treatment of HS Adalimumab is the only officially approved treatment for the management of moderate-to-severe HS by both the European Medicines Agency (EMA, June 2015) and the US Food and Drug Administration (FDA, September 2015) with a recommended dose of 160 mg sc at week 0, 80 mg sc at week 2, and then 40 mg every week starting from week 4. It is a recombinant, fully humanized, anti-tumor necrosis factor alpha (anti-TNF-α) monoclonal antibody (IgG1) that has high affinity and specificity for TNF-α. Adalimumab binds both soluble and membrane-bound forms of TNF-α and inhibits its activities by blocking its interaction with p55 and p75 cell surface TNF-α receptors.Preliminary, serendipitous evidence that anti-TNF-α agents could be effective in the management of HS came from the discipline of gastroenterology and was first reported in patients with associated Crohn's disease. [bib_ref] Maintenance infliximab for Crohn's disease: the ACCENT 1 randomised trial, Hanauer [/bib_ref] These patients were treated with anti-TNF-α agents for their Crohn's disease and simultaneously exhibited clear improvement in their HS. [bib_ref] Maintenance infliximab for Crohn's disease: the ACCENT 1 randomised trial, Hanauer [/bib_ref] [bib_ref] Treatment of hidradenitis suppurativa with anti-tumor necrosis factor-α inhibitors, Haslund [/bib_ref] Based on these observations, several studies have been conducted in order to unravel a possible role of TNF-α and other proinflammatory cytokines in the pathogenesis of HS. [bib_ref] Increased serum tumour necrosis factor-alpha in hidradenitis suppurativa patients: is there a..., Matusiak [/bib_ref] [bib_ref] Elevated levels of tumour necrosis factor (TNF)-a, interleukin (IL)-1β and IL-10 in..., Van Der Zee [/bib_ref] [bib_ref] Adalimumab (antitumour necrosis factor-α) treatment of hidradenitis suppurativa ameliorates skin inflammation: an..., Van Der Zee [/bib_ref] Moreover, case reports, series, and later clinical trials have focused on the efficacy of adalimumab and other TNF-α inhibitors in the treatment of HS. Indeed, in an in vivo study by Matusiak et al, the serum concentration of TNF-α in patients with HS was significantly higher compared with that of healthy controls, although it was not correlated with disease severity. [bib_ref] Increased serum tumour necrosis factor-alpha in hidradenitis suppurativa patients: is there a..., Matusiak [/bib_ref] In another study by van der Zee et al, the levels of TNF-α along with interleukin 1β (IL-1β) and IL-10 were elevated in lesional and perilesional skin of patients with HS compared with healthy controls and even psoriatic skin. [bib_ref] Elevated levels of tumour necrosis factor (TNF)-a, interleukin (IL)-1β and IL-10 in..., Van Der Zee [/bib_ref] These results demonstrated the strongly inflammatory nature of HS and showed that inflammation extends beyond the visibly inflamed border. [bib_ref] Elevated levels of tumour necrosis factor (TNF)-a, interleukin (IL)-1β and IL-10 in..., Van Der Zee [/bib_ref] The same author in another in situ and ex vivo study, which was part of larger placebo-controlled double-blind phase IIb trial on the efficacy and safety of adalimumab in patients with moderate-to-severe HS, showed that adalimumab treatment inhibits important cytokines (IL-1β) and inflammatory cell ## 369 Adalimumab in the treatment of hidradenitis suppurativa numbers (CD11c+ dendritic cells) in lesional skin. [bib_ref] Adalimumab (antitumour necrosis factor-α) treatment of hidradenitis suppurativa ameliorates skin inflammation: an..., Van Der Zee [/bib_ref] Also, in this study, it was evident that lack of clinical improvement highly correlated with the lack of alterations in the leucocyte subset scores and cytokine levels. [bib_ref] Adalimumab (antitumour necrosis factor-α) treatment of hidradenitis suppurativa ameliorates skin inflammation: an..., Van Der Zee [/bib_ref] ## Real life evidence about the use of adalimumab in hs -early case series and open-label studies The first data about the efficacy of adalimumab on HS came from several case series in the majority of which the drug was administered with the standard dose regimen of psoriasis (80 mg sc at week 0, 40 mg sc at week 1, and then 40 mg sc every other week). Unfortunately, in each study a different tool for the evaluation of improvement was used and this did not allow for direct comparisons between them. In general, case reports concerning 42 patients who received adalimumab for severe HS reported a cumulative response rate of 58% (≥50% in 23 patients) with a relapse rate of 71% (10 out of 14 patients). [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] [bib_ref] Efficacy of adalimumab in recalcitrant hidradenitis suppurativa, Sotiriou [/bib_ref] [bib_ref] Hidradenitis suppurativa managed with adalimumab, Yamauchi [/bib_ref] [bib_ref] Effective long-term control of refractory hidradenitis suppurativa with adalimumab after failure of..., Arenbergerova [/bib_ref] [bib_ref] Long-term successful adalimumab therapy for hidradenitis suppurativa, Blanco [/bib_ref] In the majority of patients, the drug was well tolerated. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] In a study by Arenbergerova et al, eight patients with severe HS were treated for 1 year with adalimumab in a standard psoriasis regimen and were subsequently followed for 1 year. [bib_ref] Effective long-term control of refractory hidradenitis suppurativa with adalimumab after failure of..., Arenbergerova [/bib_ref] All patients improved within 4-6 weeks. Three of them demonstrated long-lasting improvement, while five showed recurrences after a mean disease-free period of 9.5 months. [bib_ref] Effective long-term control of refractory hidradenitis suppurativa with adalimumab after failure of..., Arenbergerova [/bib_ref] Blanco et al used adalimumab in six patients with the previously mentioned dose regimen. All of the patients exhibited significant improvements in DLQI and in the number of affected areas, nodules, and fistulas. These results were maintained for a mean follow-up period of 21.5 months. [bib_ref] Long-term successful adalimumab therapy for hidradenitis suppurativa, Blanco [/bib_ref] In two open-label prospective studies, adalimumab was administered in doses higher than the approved regimen for psoriasis. Amano et al treated 10 HS patients with adalimumab for 12 weeks at doses of 160 mg sc at week 0, 80 mg sc at week 1, and the 40 mg sc every other week. [bib_ref] A prospective open-label clinical trial of adalimumab for the treatment of hidradenitis..., Amano [/bib_ref] Clinically significant improvement was not observed in any of the six patients who completed the study. [bib_ref] A prospective open-label clinical trial of adalimumab for the treatment of hidradenitis..., Amano [/bib_ref] Sotiriou et al in another open, prospective study with 15 patients suffering from moderate-to-severe HS administered adalimumab with the following dose: 80 mg sc at week 0 and then 40 mg sc every week for 24 weeks. Significant reduction in Sartorius score was obtained at week 24 with a marked improvement during the first month. [bib_ref] A prospective open-label clinical trial of efficacy of the every week administration..., Sotiriou [/bib_ref] Mean time to relapse was 11 weeks after treatment cessation, but even at the final visit, Sartorius score was lower than at baseline. 43 ## Randomized controlled trials (rcts) for the evaluation of the efficacy and safety of adalimumab in the treatment of hs early rcts Miller et al conducted a prospective, double-blind, placebocontrolled clinical trial which included 21 patients who suffered from severe HS (Hurley stage II and III) for at least 6 months. [bib_ref] A double blind placebo controlled randomized trial of adalimumab in the treatment..., Miller [/bib_ref] Fifteen patients received adalimumab (80 mg sc at baseline followed by 40 mg every other week) for 12 weeks, while seven patients received placebo (1:2 placebo/active). [bib_ref] A double blind placebo controlled randomized trial of adalimumab in the treatment..., Miller [/bib_ref] A significant reduction in Sartorius score was seen after 6 weeks and an almost significant reduction after 12 weeks of active treatment when compared with placebo (P=0.024 and P=0.07, respectively). [bib_ref] A double blind placebo controlled randomized trial of adalimumab in the treatment..., Miller [/bib_ref] Adalimumab was well tolerated, in general, but the active group experienced an almost significantly greater number of adverse events (mostly mild infections). [bib_ref] A double blind placebo controlled randomized trial of adalimumab in the treatment..., Miller [/bib_ref] Kimbal et al conducted an even larger double-blind placebo-controlled randomized trial that included 154 patients with moderate-to-severe HS who were unresponsive or intolerant to oral antibiotics. [bib_ref] Adalimumab for the treatment of moderate to severe hidradenitis suppurativa: a parallel..., Kimbal [/bib_ref] Patients were randomized to receive adalimumab (1:1:1) either 40 mg sc once weekly or 40 mg sc every other week or placebo for 16 weeks (period 1). [bib_ref] Adalimumab for the treatment of moderate to severe hidradenitis suppurativa: a parallel..., Kimbal [/bib_ref] Then all patients received adalimumab, 40 mg every other week, at the beginning of period 2, but switched to weekly dosing if the response was suboptimal (HS-PGA score of moderate or worse) at weeks 28 or 31. [bib_ref] Adalimumab for the treatment of moderate to severe hidradenitis suppurativa: a parallel..., Kimbal [/bib_ref] At week 16, clinical responses (i.e. an HS-PGA score of clear, minimal, or mild with at least a 2-grade improvement relative to baseline) were achieved by 17.6% of weekly patients, 9.6% of every-other-week patients, and 3.9% of placebo patients. [bib_ref] Adalimumab for the treatment of moderate to severe hidradenitis suppurativa: a parallel..., Kimbal [/bib_ref] A decrease in response was observed after the switch from weekly to every-other-week dosing in period 2. [bib_ref] Adalimumab for the treatment of moderate to severe hidradenitis suppurativa: a parallel..., Kimbal [/bib_ref] Headache and injection site reactions were the most common adverse events, while the serious adverse event rates were 3.9%, 5.8%, and 7.8% for placebo, every-other-week, and weekly patients, respectively. [bib_ref] Adalimumab for the treatment of moderate to severe hidradenitis suppurativa: a parallel..., Kimbal [/bib_ref] ## Pioneer study The most recent and most well-powered phase III, randomized placebo-controlled trial for the evaluation of the efficacy and safety of adalimumab in treatment of moderate-to-severe HS is the PIONEER study ( I and II). [bib_ref] Safety and efficacy of adalimumab in patients with severe hidradenitis suppurativa: results..., Kimbal [/bib_ref] [bib_ref] Safety and efficacy of adalimumab in patients with severe hidradenitis suppurativa: results..., Jemec [/bib_ref] In this study, patients who had an established diagnosis (duration ≥1 year) of moderate-to-severe HS (lesions in ≥2 distinct body areas, one of which must be Hurley II or III) Clinical, Cosmetic and Investigational Dermatology 2016:9 submit your manuscript | www.dovepress.com ## Dovepress ## Dovepress ## 370 Fotiadou et al were randomized (1:1) to receive either placebo or adalimumab 40 mg weekly for 12 weeks (period A). Specifically, the adalimumab group received 160 mg at week 0, followed by 80 mg at week 2 and 40 mg weekly from week 4 through week 12. After the initial period A, they were re-randomized to receive adalimumab either 40 mg weekly or 40 mg every other week versus placebo. Those patients who were in the placebo group during period A were randomized to receive either 40 mg weekly (PIONEER I) or placebo (PIONEER II). In total, 633 patients participated in these two studies (PIONEER I: 307, PIONEER II: 321). The primary efficacy end point, in both studies, was the proportion of patients achieving a Hidradenitis Suppurativa Clinical Response (HiSCR), defined as >50% reduction from baseline in total abscess and inflammatory nodule count, and no increase in abscess and draining fistula count. Secondary end points were the proportion of patients who achieved ≥30% reduction from baseline (partial response) and the change in modified Sartorius score from baseline to week 12, among others. In PIONEER I (period A) study, a significantly higher proportion of patients randomized to adalimumab achieved the primary efficacy end point HiSCR at week 12: adalimumab (64/153, 41.8%) versus placebo (40/154, 26.0%; P=0.003). Adverse events were comparable to placebo and consistent with the adalimumab safety profile; no new risks were identified. In PIONEER II (period B) study, HiSCR rate at week 12 was significantly higher for patients randomized to adalimumab (96/163, 58.9%) versus placebo (45/163, 27.6%; P<0.001). Statistically significant differences in the treatment were observed for all secondary end points, with more emphasis to the marked reduction in skin pain. DLQI was greatly improved in the adalimumab-treated patients and 50% of them reached the minimal clinically important difference, thus achieving a clear improvement in the quality of life.The safety profile for patients in both treatment groups was comparable. It must be pointed out that in both studies the improvement observed in the adalimumab group was evident as early as week 2, when compared to placebo. ## Comparison with other biologic agents Infliximab is a chimeric mouse/human monoclonal antibody against both the membrane-bound and soluble TNF-α. It has the longest "tradition" in the treatment of HS since the first observations about a possible role of anti-TNF-α agents in the management of HS were made in patients with inflammatory bowel disease who suffered also from HS and were treated with infliximab. [bib_ref] Maintenance infliximab for Crohn's disease: the ACCENT 1 randomised trial, Hanauer [/bib_ref] [bib_ref] Treatment of hidradenitis suppurativa with anti-tumor necrosis factor-α inhibitors, Haslund [/bib_ref] Ten cohort studies with at least four and no more than 11 patients in each study (around 73 patients in total) have been published regarding the use of infliximab in moderate-to-severe HS. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] [bib_ref] An update on hidradenitis suppurativa (part I): epidemiology, clinical aspects and definition..., Martorell [/bib_ref] [bib_ref] Treatment of hidradenitis suppurativa with biologic medications, Lee [/bib_ref] Almost all of these studies found that some of the patients improved with treatment. However, it seems that the best results were achieved with a more intensified regimen of 5 mg/kg at weeks 0, 2, 6 and monthly thereafter, compared with the infliximab treatment scheme for psoriasis. [bib_ref] Four-weekly infliximab in the treatment of severe hidradenitis suppurativa, Mortiary [/bib_ref] A recurrence rate of about 43% was observed after cessation of treatment in these case reports. [bib_ref] European S1 guideline for the treatment of hidradenitis supppurativa/acne inversa, Zoumboulis [/bib_ref] The only randomized placebo-controlled trial on the use of infliximab in the treatment of HS was the one performed by In this 8-week study, 33 patients with moderateto-severe HS were treated with infliximab 5 mg/kg at weeks 0, 2, and 6 or placebo. After the eighth week, there was a crossover and patients from the placebo group could take infliximab with the previously mentioned regimen. The outcome measure used in this study was HS Severity Index (HSSI). At week 8, 25% of patients had at least 50% reduction of HSSI from baseline, but this did not reach statistical significance versus placebo (primary efficacy outcome was not achieved). [bib_ref] Infliximab therapy for patients with moderate hidradenitis suppurativa: a randomized, double-blind, placebo..., Grant [/bib_ref] However, 20%-60% of patients, at week 8, reached a 25%-50% improvement in HSSI which was statistically significant compared with placebo. [bib_ref] Infliximab therapy for patients with moderate hidradenitis suppurativa: a randomized, double-blind, placebo..., Grant [/bib_ref] DLQI also showed considerable improvement. [bib_ref] Infliximab therapy for patients with moderate hidradenitis suppurativa: a randomized, double-blind, placebo..., Grant [/bib_ref] Recurrences occurred after discontinuation of treatment. In a retrospective comparative study with 20 HS patients (1:1), infliximab achieved a significantly greater improvement in mean Sartorius score (56%) compared with adalimumab (34%). [bib_ref] Four-weekly infliximab in the treatment of severe hidradenitis suppurativa, Mortiary [/bib_ref] Infliximab was administered at 5 mg/kg intravenously at weeks 0, 2, and 6, while adalimumab was given at 40 mg sc every other week, which is not the recommended dose for HS. [bib_ref] Four-weekly infliximab in the treatment of severe hidradenitis suppurativa, Mortiary [/bib_ref] A very recent retrospective national cohort study, which included 67 patients who received anti-TNF-α agents for HS, concluded that adalimumab seems to be more effective than infliximab and etanercept in the treatment of severe HS. [bib_ref] Antitumour necrosis factor-α for hidradenitis suppurativa: results from a national cohort study..., Sbidian [/bib_ref] Etanercept is a fusion recombinant protein, which fuses with the TNF receptor and interferes with TNF-α binding. According to several case reports, it has been administrated with a dose regimen of 25 mg sc twice weekly, in about 34 patients with encouraging outcomes. 2,53-54 However, in the only randomized placebo-controlled trial on the use of etanercept in HS (n=20 patients), the dose regimen was 50 mg sc twice weekly for 3 months and there was no significant difference compared with placebo. [bib_ref] Treatment of hidradenitis suppurativa with etanercept injection, Adams [/bib_ref] Ustekinumab (a human monoclonal antibody against the p40 subunit of IL-12/IL-23) in treatment of HS has been Clinical, Cosmetic and Investigational Dermatology 2016:9 submit your manuscript | www.dovepress.com ## Dovepress ## Dovepress ## 371 Adalimumab in the treatment of hidradenitis suppurativa described in one case report with three patients. [bib_ref] Experience with ustekinumab for the treatment of moderate to severe hidradenitis suppuratiav, Gulliver Wp [/bib_ref] The dose regimen used was 45 mg sc at weeks 0, 4, and 16. One of the three patients (33%) exhibited an improvement of ≥50%. [bib_ref] Experience with ustekinumab for the treatment of moderate to severe hidradenitis suppuratiav, Gulliver Wp [/bib_ref] However, it is too early to decisively conclude about the effectiveness of this biologic agent in psoriasis. Anakinra is a recombinant form of the human IL-1 receptor antagonist that inhibits the biologic activity of IL-1α and IL-1β. It has been tried in several cases of severe HS. Initial sporadic case reports have produced contradictory outcomes, while an open-label study by Leslie et al has shown a satisfactory response (i.e. decrease of Sartorius score) in five out of six patients. [bib_ref] Failure of Anakinra in a case of severe hidradenitis suppurativa, Russo [/bib_ref] [bib_ref] Two cases of severe hidradenitis suppurativa with failure of anakinra therapy, Menis [/bib_ref] [bib_ref] Successful treatment of severe hidradenitis suppurativa with anakinra, Zarchi [/bib_ref] [bib_ref] An openlabel study of anakinra for the treatment of moderate to severe..., Leslie [/bib_ref] Recently, Tzanetakou et al conducted a double-blind, randomized, placebo-controlled clinical trial with a very promising outcome. [bib_ref] Safety and efficacy of anakinra in severe hidradenitis suppurativa: a randomized clinical..., Tzanetakou [/bib_ref] It included 10 patients who received once-daily subcutaneous injection of anakinra 100 mg for 12 weeks and 10 controls. An HiSCR was achieved by 78% of patients in the anakinra group and by only 20% of patients in the control group. However, larger studies are needed in order to confirm these results. # Conclusion In the era of evidence-based medicine and the new understanding of the inflammatory pathways that characterize HS, adalimumab, to date, holds the most robust data regarding efficacy in the treatment of the disease. Apparently, adalimumab can contribute to the optimal control of the inflammatory burden of the disease and hopefully in a second level could prevent scarring. However, future larger registry-based studies are needed to further establish the efficacy and safety profile of this anti-TNF-α agent in HS.

Serum biomarkers confirming stable remission in inflammatory bowel disease Crohn's disease (CD) and ulcerative colitis (UC) have a chronic-remittent course. Optimal management of inflammatory bowel diseases (IBD) relies on early intervention, treat-to-target strategies and a tight disease control. However, it is challenging to assess the risk of relapses in individual patients. We investigated blood-based biomarkers for the confirmation of disease remission in patients with IBD. We retrospectively analyzed samples of 40 IBD patients (30 UC, 10 CD) enrolled in a tightcontrol follow-up study. Half of the patients had a flare during follow up. Serum was analyzed for S100A12 as well as S100A8/A9 and for 50 further biomarkers in a bead-based multiplex assay. The concentrations of 9 cytokines/chemokines and S100A8/A9 significantly differed in IBD patients with unstable remission (before flares) when compared to IBD patients with stable remission. Although the number of patients was small, ROC curve analyses revealed a number of biomarkers (IL-1β, IL-1RA, IL-8, IL13, IL-15, IL-21, IL-25, IFN-β, CXCL9, CXCL10, CXCL11, Galectin-1, G-CSF and S100A8/A9) that were elevated in patients with later occurring relapses. While earlier studies on peripheral biomarkers in IBD are limited to only few analytes, our study using a broad screening approach identified serum biomarkers with the potential to indicate unstable disease control in IBD, which may help to steer individual therapies to maintain remission.OPEN The management of patients with inflammatory bowel disease (IBD) is evolving. The traditional concept of a stepup therapy has been challenged and treat-to-target strategies have been proposed [bib_ref] Treat to target: A proposed new paradigm for the management of Crohn's..., Bouguen [/bib_ref]. The main treatment target is to induce and maintain disease remission, which means a control of intestinal inflammation, a normalization of life, and the prevention of long-term damage [bib_ref] Inflammatory bowel disease: Clinical aspects and established and evolving therapies, Baumgart [/bib_ref] [bib_ref] World Gastroenterology Organization Practice Guidelines for the diagnosis and management of IBD..., Bernstein [/bib_ref] [bib_ref] AGA clinical practice guidelines on the management of moderate to severe ulcerative..., Feuerstein [/bib_ref] [bib_ref] A treat-to-target update in ulcerative colitis: A systematic review, Ungaro [/bib_ref]. Both for Crohn's disease (CD) and ulcerative colitis (UC), effective biological drugs enable improved therapeutic outcomes [bib_ref] Higher trough vedolizumab concentrations during maintenance therapy are associated with corticosteroid-free remission..., Ungaro [/bib_ref]. Disease remission can be defined by endoscopic endpoints such as mucosal healing. Yet, radiologic status, patient reported outcomes, and use of non-invasive biomarkers are also conceivable measures of the therapeutic target [bib_ref] Selecting therapeutic targets in inflammatory bowel disease (STRIDE): Determining therapeutic goals for..., Peyrin-Biroulet [/bib_ref] [bib_ref] Mucosal healing as a target of therapy for colonic inflammatory bowel disease..., Walsh [/bib_ref]. Follow-up recommendations for patients with IBD based on treat-to-target strategies mainly focus on the initial treatment phase, when therapies are started in patients with active disease to induce remission 4 . However, patient follow-up upon successful initial treatment is less clear. It is important to maintain a sustained remission. Since CD and UC are both chronic-remittent diseases, quiescent phases may be followed by (seemingly unprovoked) relapsing disease. Therefore, monitoring of disease activity is the mainstay of clinical decision-making. At present, accurate monitoring of intestinal inflammation relies upon clinical indices (based upon symptoms and clinical examination) and endoscopy, in conjunction with histological investigation and imaging techniques. However, these diagnostic options have a number of drawbacks, as they are time consuming, costly, invasive and/ or not necessarily objective. Indirect, yet reliable, measures of biological disease activity are of utmost importance. Blood tests, including C-reactive protein (CRP) and erythrocyte sedimentation rate (ESR), are in common use but have insufficient sensitivity and specificity for intestinal inflammation [bib_ref] Fecal calprotectin in predicting relapse of inflammatory bowel diseases: A meta-analysis of..., Mao [/bib_ref]. Currently there are no means to predict the long-term disease course, and adjusting treatment to the actual needs of patients is especially difficult when the patient is feeling well. In these phases, invasive measures of subclinical disease activity such as endoscopy are often not considered acceptable [bib_ref] Do we still need predictors of disease severity when applying a treat-to-target..., Louis [/bib_ref] www.nature.com/scientificreports/ biomarkers would thus be helpful in evaluating the risk for relapses [bib_ref] Approaches to integrating biomarkers into clinical trials and care pathways as targets..., Dulai [/bib_ref] [bib_ref] ECCO-ESGAR guideline for diagnostic assessment in IBD part 1: Initial diagnosis, monitoring..., Maaser [/bib_ref] [bib_ref] ECCO-ESGAR guideline for diagnostic assessment in IBD part 2: IBD scores and..., Sturm [/bib_ref]. Even though some biomarkers measured in blood or stool have been shown to offer variable degree of utility in monitoring gastrointestinal tract inflammation in IBD, in clinical practice there is still an unmet need for biomarkers that could assess the stability of disease remission and the risk of relapse [bib_ref] Challenges in IBD research: Precision medicine, Denson [/bib_ref] [bib_ref] Combined use of common fecal and blood markers for detection of endoscopically..., Mak [/bib_ref] [bib_ref] Utility of surrogate markers for the prediction of relapses in inflammatory bowel..., Musci [/bib_ref] [bib_ref] Serum calprotectin as a biomarker for Crohn's disease, Meuwis [/bib_ref]. The effect of tight control management on Crohn's disease (CALM) trial has demonstrated that treatment escalation based on symptoms combined with elevated serum CRP and/ or fecal calprotectin was better than symptom-based escalation alone [bib_ref] Effect of tight control management on Crohn's disease (CALM): A multicentre, randomised, Colombel [/bib_ref]. Despite a robust diagnostic accuracy, the use of fecal markers is somewhat difficult in everyday practice especially from the patients' perspective [bib_ref] Comparative acceptability and perceived clinical utility of monitoring tools: A nationwide survey..., Buisson [/bib_ref] [bib_ref] Patients' perceptions of faecal calprotectin testing in inflammatory bowel disease: Results from..., Kalla [/bib_ref]. Consequently, stool markers are actually monitored in a minority of patients [bib_ref] US practice patterns and impact of monitoring for mucosal inflammation after biologic..., Limketkai [/bib_ref] [bib_ref] A nationwide 2010-2012 analysis of U.S. health care utilization in inflammatory bowel..., Van Deen [/bib_ref]. Patients with IBD prefer bloodbased over fecal biomarkers [bib_ref] Compliance with the faecal calprotectin test in patients with inflammatory bowel disease, Marechal [/bib_ref]. However, blood-based bio-markers have shown poor accuracy [bib_ref] C-reactive protein, fecal calprotectin, and stool lactoferrin for detection of endoscopic activity..., Mosli [/bib_ref]. A need therefore exists for blood-based biomarkers that accurately detect disease activity in IBD. In a previously published prospective 36-month multicenter study, we demonstrated the utility of fecal biomarkers to predict the flare risk in IBD patients after reaching inactive disease [bib_ref] Improving relapse prediction in inflammatory bowel disease by neutrophil-derived S100A12, Dabritz [/bib_ref] [bib_ref] Granulocyte macrophage colony-stimulating factor auto-antibodies and disease relapse in inflammatory bowel disease, Dabritz [/bib_ref]. Time course analysis of S100A12 up to 9 months before and after relapse showed a clear increase of fecal but not serum S100A12 concentrations up to 6 months before clinical relapse. We now extend our work to candidate biomarkers that appear potentially related to inflammatory processes in IBD and can be analyzed in serum samples collected during remission. We aimed at identifying biomarker panels to identify patients who may need an optimized and/or intensified maintenance therapy to avoid disease flares. # Materials and methods Patients and study design. In a prospective multicenter study, patients with IBD in remission were consecutively recruited and followed up between April 2008 and June 2011 in four independent German outpatient specialized clinics as previously described [bib_ref] Improving relapse prediction in inflammatory bowel disease by neutrophil-derived S100A12, Dabritz [/bib_ref] [bib_ref] Granulocyte macrophage colony-stimulating factor auto-antibodies and disease relapse in inflammatory bowel disease, Dabritz [/bib_ref]. The diagnoses of CD and UC were confirmed as described previously [bib_ref] Improving relapse prediction in inflammatory bowel disease by neutrophil-derived S100A12, Dabritz [/bib_ref] [bib_ref] Granulocyte macrophage colony-stimulating factor auto-antibodies and disease relapse in inflammatory bowel disease, Dabritz [/bib_ref]. Patients with coexisting and serious cardiopulmonary, hepatic, renal, neurologic, psychiatric, and rheumatologic disease, a history of HIV and/or hepatitis B and C were excluded from the study. Patients were assessed at a minimum of 3-month intervals or when relapse occurred. Serum and stool samples were prospectively collected at each visit when available. In addition to baseline characteristics, symptoms, medication, clinical signs, and standard laboratory results (full blood count, ESR, CRP) were recorded throughout the study. For the present analyses, 80 serum samples were retrospectively selected from 40 IBD patients based on the occurrence of disease flares at follow-up within a maximum of 1 year. For each patient paired samples either during an initial (visit 1) or a follow-up visit (visit 2) were available, making up 60 samples from 30 UC patients and 20 samples from 10 CD [fig_ref] Table 1: Characteristics of included IBD patients [/fig_ref]. Half of patients in both groups either remained in stable remission (experiencing remission both at visit 1 and 2) or were classified as unstable remission (with remission at visit 1, but an acute Assessment of disease activity. Disease activity was assessed based on the Crohn's disease activity index (CDAI) for patients with CD and the ulcerative colitis activity index (UCAI) for patients with UC. Remission was defined as a CDAI < 150 or UCAI < 5. Relapse was defined as follows: CDAI > 250 over 2 consecutive weeks or a CDAI > 150 with an at least 70-points of increase within 2 weeks as compared with CDAI at the previous study visit; UCAI > 6 over 2 consecutive weeks or a UCAI > 4 with an at least 3-points of increase within 2 weeks as compared with UCAI at the previous study visit. Immunoassays. Concentrations of S100A12 were determined by a double-sandwich ELISA, as described previously [bib_ref] Neutrophil derived human S100A12 (EN-RAGE) is strongly expressed during chronic active inflammatory..., Foell [/bib_ref] [bib_ref] Faecal S100A12 as a non-invasive marker distinguishing inflammatory bowel disease from irritable..., Kaiser [/bib_ref]. Calprotectin (S100A8/A9) was measured by a commercial sandwich ELISA (Bühlmann Laboratories AG, Schoenenbuch, Switzerland). Validated multiplexed immunoassays were used to measure 50 analytes using Luminex xMAP proteomics technology (Austin TX, USA). Fifty different carboxylated magnetic beads, each with a distinct emitting fluorescence pattern, were purchased from Luminex Corporation (Austin, TX, USA). Capture antibodies (commercially purchased) for 50 analytes were covalently coupled to the microspheres as described previously [bib_ref] Blood and synovial fluid cytokine signatures in patients with juvenile idiopathic arthritis:..., De Jager [/bib_ref] [bib_ref] Improved multiplex immunoassay performance in human plasma and synovial fluid following removal..., De Jager [/bib_ref] [bib_ref] Simultaneous detection of 15 human cytokines in a single sample of stimulated..., De Jager [/bib_ref]. Acquisition was performed with a BioRad FlexMAP3D (BioRad laboratories, Hercules, USA) in combination with xPONENT software, version 4.2 (Luminex). Data were analyzed by 5-parametric curve fitting using Bio-Plex Manager software, version 6. Kruskal-Wallis test with Bonferroni post-hoc analyses were applied to correct for multiple comparisons. Inferential statistics were intended to be exploratory, not confirmatory, and were interpreted accordingly. Receiver operated characteristics (ROC) curve analyses (GraphPad Prism 8.0) were applied to test for the prediction of flares and the distinction of populations at risk (stable or unstable remission groups), with calculation of the area under curve (AUC). Binary logistic regression analyses were performed to test for multiparametric prediction models. The significance level was set at P < 0.05 and confidence levels at 95%. # Results Our broad serum biomarker analyses in IBD followed a specific methodology: a first sample was available initially when the patients were recruited, and all patients were in disease remission at this time point [fig_ref] Table 1: Characteristics of included IBD patients [/fig_ref] Within the IBD cohort, the acquired serum level data of 52 analytes did not result in specific grouping of patients when subjected to unsupervised clustering analyses [fig_ref] Figure 1: Analysis of inflammatory parameters and multiplexed serum markers in IBD [/fig_ref] , but yielded 9 markers with concentrations that differed in patients with future unstable remission compared to those with future stable remission (i.e. without subsequent relapse): already at the baseline visit (T1) serum levels of IL-1β, IL-15, IL-18, IL-21, IL-25, IFN-β, CXCL9, CXCL10 and S100A8/A9 were higher in those who later experienced disease relapse [fig_ref] Figure 1: Analysis of inflammatory parameters and multiplexed serum markers in IBD [/fig_ref] , [fig_ref] Table 2: Performance of biomarkers showing significant differences [/fig_ref]. For the median values of S100A8/A9 (p = 0.016) and CXCL9 (p = 0.031) the statistical difference was confirmed in post-hoc Bonferroni corrections for multiple comparisons [fig_ref] Table 2: Performance of biomarkers showing significant differences [/fig_ref]. Standard laboratory markers (Hb, white blood cell counts, and CRP; [fig_ref] Table 1: Characteristics of included IBD patients [/fig_ref] in the group with future IBD relapse did not significantly differ from those in the future non-relapse group. In addition, fecal markers of inflammation (fecal calprotectin and S100A12, respectively) revealed no significant differences between the patient groups [fig_ref] Table 1: Characteristics of included IBD patients [/fig_ref]. In contrast to the differences in baseline levels and except for CXCL10, there was no clear trend when comparing the T1 with T2 samples. The differences between inactive and active disease appear rather small in comparison with the differences observed with background activity at inclusion [fig_ref] Figure 1: Analysis of inflammatory parameters and multiplexed serum markers in IBD [/fig_ref]. In patients with unstable remission, experiencing a future flare, multiple correlation analyses of significantly different serum markers in IBD T1-samples [fig_ref] Figure 1: Analysis of inflammatory parameters and multiplexed serum markers in IBD [/fig_ref] as well as routine blood and fecal markers of inflammation [fig_ref] Table 1: Characteristics of included IBD patients [/fig_ref] ; Supplementary Table S1) revealed that mainly markers which can be linked to T cell activation (IL-15, IL-18, IL-21, IL-25) or IFNγ-signaling (CXCL9) but also IL-1β and IFNβ cluster together in positive association [fig_ref] Figure 2: Multiple correlation analyses of inflammation biomarkers in IBD [/fig_ref]. This pattern only marginally differs from that observed in patients remaining in stable remission. Serum and inflammatory marker associations in these patients with stable remission predominantly reveal marked negative correlations with fecal calprotectin, which is inverse to what we observed with respect to fecal S100A12 [fig_ref] Figure 2: Multiple correlation analyses of inflammation biomarkers in IBD [/fig_ref]. Restricting our analyses to UC patients [fig_ref] Figure 3: Analysis of inflammatory parameters and multiplexed serum markers in UC [/fig_ref] did not benefit the overall unsupervised clustering based on the acquired levels of 52 serum markers [fig_ref] Figure 3: Analysis of inflammatory parameters and multiplexed serum markers in UC [/fig_ref] , but we observed that concentrations of 14 analytes (IL-1β, IL-1RA, IL-8, IL-13, IL-15, IL-21, IL-25, IFN-β, CXCL9, CXCL10, CXCL11, S100A8/A9, G-CSF and Galectin-1) were significantly higher in samples from patients with unstable remission compared to patients with stable remission [fig_ref] Figure 2: Multiple correlation analyses of inflammation biomarkers in IBD [/fig_ref]. For the median values of S100A8/A9 (p < 0.001), Galectin-1 (p = 0.002), CXCL11 [fig_ref] Table 2: Performance of biomarkers showing significant differences [/fig_ref]. Paralleling observations in the total IBD cohort, standard blood or fecal markers of inflammation (Hb, white blood cell counts, CRP, fecal calprotectin, fecal S100A12) in patients with future relapse did not significantly differ from those with future stable remission [fig_ref] Table 1: Characteristics of included IBD patients [/fig_ref]. When comparing biomarker levels in T1 with T2 samples we only observed significant differences in CXCL10 and CXCL11 [fig_ref] Figure 3: Analysis of inflammatory parameters and multiplexed serum markers in UC [/fig_ref]. Most patients (n = 27) within the UC cohort were seen within less than 60 days following T1 for their respective follow-up T2 visit. When excluding three patients with T2 visits > 100 days from our data set, this did not result in major changes among the identified markers with significantly different serum levels with respect to future flare or stable remission [fig_ref] Table 2: Performance of biomarkers showing significant differences [/fig_ref]. When subjecting only data acquired from UC patients to multiple correlation analyses, the picture marginally differs from what we observed in total IBD. Both among patients with future relapse or stable remission we observed mainly markers linked to T cell activation (IL-15, IL-18, IL-21, IL-25) or IFNγ-signaling (CXCL9, CXCL11) but also IL-1β and IFNβ to cluster together in positive association [fig_ref] Figure 4: Multiple correlation analyses of inflammation biomarkers in UC [/fig_ref]. Associations of serum cytokines with IL-8, G-CSF, CXCL10, S100A8/A9 and Galectin-1 appear to mainly differ in T1-samples obtained from patients remaining in remission or experiencing a future flare. Further, as observed in the total IBD cohort, serum and inflammatory marker associations in patients with future stable remission predominantly reveal marked negative correlations with fecal calprotectin, which is inverse to what we observed with respect to fecal S100A12 [fig_ref] Figure 4: Multiple correlation analyses of inflammation biomarkers in UC [/fig_ref]. Prompted by ROC analyses revealing differences between patients with future stable versus unstable remission for both the whole IBD and the UC cohort [fig_ref] Table 2: Performance of biomarkers showing significant differences [/fig_ref] , we evaluated predictive models taking the significant biomarkers into account. The analyses have to be interpreted with care due to the balance of patient samples and analytes and the limitations of multiple comparisons. Binary nominal logistic regression analyses showed that S100A8/A9 has a predictive power for all IBD patients and even better for UC patients. In the latter group, a model adding S100A8/A9 to measurements of CXCL11 yields a predictive power of 80% [fig_ref] Table 2: Performance of biomarkers showing significant differences [/fig_ref]. In UC patients, both CXCL11 (sensitivity 67%, specificity 87%, likelihood ration 5.0) and S100A8/A9 (sensitivity 73%, specificity 87%, likelihood ration 5.5) could be confirmed as markers differentiating patients with future flares from those with stable remission . # Discussion While it is unlikely that serum biomarkers will ever replace invasive tests, such as endoscopy, they could be useful as inflammatory markers filtering for the need of invasive investigations while monitoring the patients' disease course. We extend our previous work that has revealed a predictive power of fecal S100A12 and calprotectin, but only a weak association of their serum levels with flare risk in IBD patients. For further analyses of candidate blood-based markers, we created a cohort of patients who either remained in remission during follow up ("stable remission" group) or who consecutively experienced a relapse in a predefined time period ("unstable remission" group). We present 16 biomarkers with the potential to indicate unstable remission in IBD. In particular, 14 molecular markers with elevated values in UC patients with unstable remission were identified. Those can be www.nature.com/scientificreports/ indicative of a background T(h1) cell activation, but also innate immune activation (e.g., shown by S100A8/ A9) that is more pronounced in those considered in clinical remission who likely continue having subclinical inflammatory processes. There were only relatively small alterations in the biomarker concentrations when comparing T1 and T2 samples, both in patients with stable and unstable remission. It appears conceivable that the fluctuations in serum biomarkers during inactive and active disease phases in individual patients are less prominent than dysbalanced immune activity that is present as a background characteristic of the patient group, as these differences are even observed during times of clinically inactive disease in remission. Although our results have to be interpreted www.nature.com/scientificreports/ with caution in light of the limited patient numbers, the correlograms indicate that differences in T cell activation and to some extent also innate immunity as well as IFN-related pathways may influence a background immune activation that can influence the risk of relapsing disease. A set of markers used to indicate molecular signatures may be more suitable for precision medicine than single biomarkers, as they can unmask complex processes rather than a single phenomenon. For patients with Rheumatoid Arthritis (RA), treat-to-target strategies and tight control are nowadays cornerstones of patient management, and biomarker panels have been introduced and validated for clinical purposes [bib_ref] Pretreatment multi-biomarker disease activity score and radiographic progression in early RA: Results..., Hambardzumyan [/bib_ref]. There are important distinctions between RA, CD and UC, suggesting differences in the underlying pathways driving each disease. However, the unifying treatment target is disease remission, mainly defined as clinical remission supported by endoscopy or imaging. Biomarker remission (normal blood cells counts, CRP, fecal calprotectin) is considered as an adjunctive target 3 . Although fecal markers are considered non-invasive means to monitor intestinal inflammation, the lack of conclusive data on relapse prediction and the low acceptance of stool sampling by patients is limiting their widespread use [bib_ref] Comparative acceptability and perceived clinical utility of monitoring tools: A nationwide survey..., Buisson [/bib_ref] [bib_ref] Patients' perceptions of faecal calprotectin testing in inflammatory bowel disease: Results from..., Kalla [/bib_ref] [bib_ref] US practice patterns and impact of monitoring for mucosal inflammation after biologic..., Limketkai [/bib_ref] [bib_ref] A nationwide 2010-2012 analysis of U.S. health care utilization in inflammatory bowel..., Van Deen [/bib_ref] [bib_ref] Compliance with the faecal calprotectin test in patients with inflammatory bowel disease, Marechal [/bib_ref]. A need therefore exists for blood-based biomarkers that accurately detect disease activity in IBD. A recent study by D'Haens et al. used a commercial assay of multiple markers indicating mucosal damage and repair processes (PROMETHEUS Monitr Crohn's Disease Test) to calculate an endoscopic healing index (EHI) that identifies patients with resolution of endoscopic disease activity [bib_ref] Mucosal healing at 3 months predicts long-term endoscopic remission in anti-TNF-treated luminal..., Af Bjorkesten [/bib_ref]. The test applies a proprietary algorithm with 13 biomarkers to produce a quantitative EHI score. The authors used very strong outcome measures of endoscopic healing that were not available to us. The study also didn't test the prediction of remission of risk or relapses. Some of the biomarkers that revealed promising results in our small IBD cohort were not considered by D'Haens et al., because they showed poor analytical reproducibility, low detection rate, and/or lack of correlation to disease severity in preliminary studies. These markers were eliminated from further consideration, and data are not presented. The study was funded by Prometheus, several authors were either employees or at least had a relation to Prometheus, and the company was responsible for running assays and analyses that are only partially reported [bib_ref] Development and validation of a test to monitor endoscopic activity in patients..., D&apos;haens [/bib_ref]. Even though this limits the interpretation of data, it appears promising that the accuracy of the blood-based multi-marker set was comparable to fecal calprotectin and better than measurement of serum CRP. As a limitation, we could only use clinical disease activity indices (CDAI and UCAI) to define stable and unstable remission. Endoscopic or histological disease activity measures were not available from the participants included in remission. It is a drawback of CDAI/UCAI scores that they may not correlate well with endoscopically proven intestinal inflammation. The relatively small sample number limits the statistical power of our study. Especially the number of samples from CD patients available for the retrospective project is a significant limitation. This also excluded further stratification of patients, e.g. with regard to disease characteristics or therapies. As an example, two CD patients received systemic steroids at inclusion, one reduced the dose at visit 1 from 5 to 4 mg/day, and the other one actually stopped as visit 1. We cannot fully exclude that medication changes in therapy influence the risk of flare at future time point due to poorly controlled disease. In addition, we could not correlate our data to repeated endoscopic measures in the cohort recruited in disease remission. However, we consider the results promising and in line with other studies showing that serum biomarker panels have a potential to identify IBD from symptomatic controls and to predict future disease course [bib_ref] Serum proteomic profiling at diagnosis predicts clinical course, and need for intensification..., Kalla [/bib_ref]. Future studies will need to confirm whether elevated inflammatory markers in IBD patients in clinical remission as defined by clinical disease activity indices may represent a stage of residual inflammation, which progresses to cause an eventual clinical relapse of the disease. Conversely, it is conceivable that measuring biomarker panels may serve as a tool for measuring the effects of treatment. Consequently, treatment of IBD could be tapered at a point where the biomarkers suggest that the relapse of disease is unlikely to occur within a defined period. Our analyses indicate that especially S100A8/A9 analyses may have a predictive power for all IBD patients and even better for UC patients. A recent other study also suggested S100A8/A9 and either CRP or albumin for a prognostic model to predict treatment escalation in IBD [bib_ref] Serum calprotectin: A novel diagnostic and prognostic marker in inflammatory bowel diseases, Kalla [/bib_ref]. In conclusion, there is a strong need for defining appropriate variables for follow-up recommendations which is vital to IBD treatment and management. Based on our results, it seems a feasible goal to apply molecular signatures, measurable in blood and available for tight monitoring of disease activity. Future studies will test the treat-to-target approaches in larger cohorts and can be used to validate the usefulness of biomarker signatures for tight monitoring of disease activity. [fig] Figure 1: Analysis of inflammatory parameters and multiplexed serum markers in IBD. (A) Illustration of the experimental layout. IBD patients during remission were included into the study (T1). Patients were followed-up over 1 year. Half of the patients had a relapse during follow-up. A second sample was obtained at T2, either at the time of the flare of at the end of follow-up in remission. (B) Heatmap of serum marker data from bead array assay, ELISA data (S100A8/A9, S100A12) and routine inflammatory parameters (ESR, CRP, WBC) following unsupervised hierarchical clustering and complete linkage analyses (RStudio R 3.5.0, the R Foundation for Statistical Computing, Vienna, Austria). Red and green indicate future relapse or stable remission of UC patients, light red and light green indicate future relapse or stable remission of CD patients. (C) Markers with significantly different levels at T1 between IBD patients with stable remission (stable R) or unstable remission (unstable R) (left panel column), and comparison of respective marker levels between T1 and T2 during stable (middle panel column) or unstable remission (right panel column). Acquisition was performed with a BioRad FlexMAP3D (BioRad laboratories, Hercules, USA) in combination with xPONENT software, version 4.2 (Luminex). Data were analyzed using Bio-Plex Manager software, version 6.1.1 (BioRad). Data of individual serum biomarkers were analyzed by Mann-Whitney U or, when paired, by Wilcoxon signed rank test. 008), CXCL9 (p = 0.012), CXCL10 (p = 0.024), and IL-21 (p = 0.038) the statistical difference was confirmed in post-hoc Bonferroni corrections for multiple comparisons [/fig] [fig] Figure 2: Multiple correlation analyses of inflammation biomarkers in IBD. Serum markers with significantly different levels at T1 based on whether experiencing future flare or remaining in remission (as inFig. 1C) as well as routine clinical laboratory (WBC, ESR, CRP) and fecal markers of inflammation were analyzed for their association based on spearman rank (RStudio, R 3.5.0, the R Foundation for Statistical Computing, Vienna, Austria). [/fig] [fig] Figure 3: Analysis of inflammatory parameters and multiplexed serum markers in UC. (A) Illustration of the experimental layout, restricted to UC patients during remission at inclusion (T1). Patients were followed-up over 1 year. Half of the patients had a relapse during follow-up. A second sample was obtained at T2, either at the time of the flare of at the end of follow-up in remission. (B) Heatmap of serum marker data from bead array assay, ELISA data (S100A8/A9, S100A12) and routine inflammatory parameters (ESR, CRP, WBC) following unsupervised hierarchical clustering and complete linkage analyses (RStudio, R 3.5.0, the R Foundation for Statistical Computing, Vienna, Austria). (C) Markers with significantly different levels at T1 between IBD patients with stable remission (stable R) or unstable remission (unstable R) (left panel column), and comparison of respective marker levels between T1 and T2 during stable (middle panel column) or unstable remission (right panel column). Acquisition was performed with a BioRad FlexMAP3D (BioRad laboratories, Hercules, USA) in combination with xPONENT software, version 4.2 (Luminex). Data were analyzed using Bio-Plex Manager software, version 6.1.1 (BioRad). Data of individual serum biomarkers were analyzed by Mann-Whitney U or, when paired, by Wilcoxon signed rank test. [/fig] [fig] Figure 4: Multiple correlation analyses of inflammation biomarkers in UC. Serum markers with significantly different levels at T1 based on whether experiencing future flare or remaining in remission (as inFig. 3C) as well as routine clinical laboratory (WBC, ESR, CRP) and fecal markers of inflammation were analyzed for their association based on spearman rank (RStudio, R 3.5.0, the R Foundation for Statistical Computing, Vienna, Austria). [/fig] [table] Table 1: Characteristics of included IBD patients. IBD inflammatory bowel disease, UC ulcerative colitis, CD Crohn's disease, TNF tumor necrosis factor, Hb hemoglobulin, WBC white blood cells, CRP C-reactive protein. and written informed consent was obtained from all patients. The authors confirm that all experiments were performed in accordance with relevant guidelines and regulations. [/table] [table] Table 2: Performance of biomarkers showing significant differences (all in pg/ml except IFNβ in U/ml and S100A8/A9 in ng/ml). CI confidence interval, AUC area under curve; a significance of ROC analyses; b adjusted significance using Kruskal Wallis test with Bonferroni post-hoc correction for multiple comparisons. Significant P-values indicated in bold. [/table]

CITED2 Mutation and methylation in children with congenital heart disease Background: The occurrence of Congenital Heart Disease (CHD) is resulted from either genetic or environmental factors or the both. The CITED2 gene deletion or mutation is associated with the development of cardiac malformations. In this study, we have investigated the role of CITED2 gene mutation and methylation in the development of Congenital Heart Disease in pediatric patients in China. Results: We have screened 120 pediatric patients with congenital heart disease. Among these patients, 4 cases were detected to carry various CITED2 gene heterozygous mutations (c.550G > A, c.574A > G, c.573-578del6) leading correspondingly to the alterations of amino acid sequences in Gly184Ser, Ser192Gly, and Ser192fs, respectively. No CITED2 gene mutations were detected in the control group. At the same time, we found that CITED2 mutations could inhibit TFAP2c expression. In addition, we have demonstrated that abnormal CITED2 gene methylation was detected in most of the tested pediatric patients with CHD, which leads to a decrease of CITED2 transcription activities.Conclusions: Our study suggests that CITED2 gene mutations and methylation may play an important role in the development of pediatric congenital heart disease. # Background Congenital Heart Disease (CHD) is the most common disease in children with congenital birth defects, which is a challenge in pediatric cardiology. A body of studies has demonstrated that the congenital heart diseases are caused by either genetic or environmental factors or both. More and more gene mutations were found to be associated with CHD, such as NKX2.5, TBX5, TFAP2B, GATA-4 [bib_ref] Genetic analysis of essential cardiac transcription factors in 256 patients with non-syndromic, Kodo [/bib_ref] [bib_ref] Analyses of GATA4, NKX2.5, and TFAP2B genes in subjects from southern China..., Xiong [/bib_ref]. Although gene mutations are potential causes for the development of CHD [bib_ref] Congenital heart disease: current knowledge about causes and Inheritance, Blue [/bib_ref] , various environmental factors that affect mother during the pregnancy also play an important role in promoting and developing of CHD. For example, epigenetic modifications and gene methylation changes in fetus during pregnancy can affect gene expression levels during the development [bib_ref] Maternal genome-wide DNA methylation patterns and congenital heart defects, Chowdhury [/bib_ref] [bib_ref] Genome-wide survey reveals dynamic widespread tissue-specific changes in DNA methylation during development, Liang [/bib_ref] [bib_ref] Screening for differential methylation status in fetal myocardial tissue samples with ventricular..., Zhu [/bib_ref]. Aberrant methylation in the promoter region of genes can inhibit gene transcription activities by preventing the binding of transcription factor from the target genes, which is one of the potential causes of gene silence and disease [bib_ref] Promoter hypermethylation-induced transcriptional down-regulation of the geneMYCT1 in laryngeal squamous cell carcinoma, Yang [/bib_ref]. CREB-binding protein (CBP)/P300-interacting transactivator 2 is a protein with ED-rich tail that in human is encoded by the CITED2 gene. CITED2 expression is regulated by a variety of factors such as hypoxia, cytokines, oxidative stress, etc. [bib_ref] A cell-autonomous role of Cited2 in controlling myocardial and coronary vascular development, Macdonald [/bib_ref]. There are a plurality of transcription factor binding sites in the promoter region of CITED2 gene, such as HIF-1, AP-2, SP1 etc., which play a vital role in CITED2 expression. Three consecutive sequence of "ACGTG" in CITED2 promoter region can maintain the stability of the combination between CITED2 and HIF-1 [bib_ref] Molecular cloning and chromosomal localization ofthe human CITED2 gene encoding p35srj/Mrg1, Leung [/bib_ref] [bib_ref] MRG1 Expression in fibroblasts is regulated by Sp1/Sp3 and an Ets transcription..., Han [/bib_ref]. HIF-1 consists of α, β two subtypes, in which HIF-1α is degraded under normal oxygen conditions and cannot be detected. However, HIF-1α can be detected in hypoxic conditions [bib_ref] Differential regulation of human PlGF gene expression inTrophoblast and nontrophoblast cells by..., Gobble [/bib_ref]. CITED2 blockes HIF-1α transcriptional activity by competitively inhibiting the interaction between HIF-1α and CBP/P300, Dysfunction of HIF-1α in CITED2 −/− mice may cause the cardiac malformation [bib_ref] The essential role of Cited2, a negative regulator for HIF-1 in heart..., Yin [/bib_ref] [bib_ref] HIF-1 and its antagonist Cited2, Du [/bib_ref]. CITED2 also functions as a transcriptional co-activator by recruiting the combination of CBP/P300 and TFAP2. Deficiencies in TFAP2 co-activation have been suggested to cause laterality defect in CITED2 -/mice [bib_ref] Cardiacmalformations, adrenalagenesis, neural crest defects and exencephaly in mice lacking Cited2, a..., Bamforth [/bib_ref]. CITED2 gene mutation was first reported by Sperling et al. in congenital heart disease [bib_ref] Identification and functional analysis of CITED2 mutations in patients with congenital heart..., Sperling [/bib_ref] and the mutation could significantly diminish TFAP2c co-activation. However, the relationship between CITED2 mutations and CHD as well as the epigenetic modification of the gene is not clear. In the present study, we have screened 120 pediatric patients with CHD and tried to reveal the role of gene mutations and epigenetic modifications in the development of CHD in children. Our study suggests that CITED2 gene mutations and methylation may play an important role in the development of pediatric congenital heart disease. # Methods ## Subjects Blood samples were obtained from 120 pediatric patients (from 8 days to 18 years) and from 100 normal children with matched age. In addition, myocardial tissues were collected from 31 pediatric patients with CHD in Children's Hospital of Chongqing Medical University and 2 normal cardiac tissue samples were obtained from children died of accident [fig_ref] Table 1: Phenotypies and clinical manifestations of CHD in pediatric patients [/fig_ref]. The protocols and assays of the study was approved by the Ethics Committee in Children's Hospital Chongqing Medical University before the start of the study. ## Reagents Blood and Tissue DNA extraction kit was purchased from Tiangen; plasmid pEGFP-C1 was purchased from Dingguo biotechnology company; LA Tag enzyme, pMD19-T simple plasmid restriction enzyme ScaI and KpnI, DNA ligase, DNA marker, Reverse transcription kits were purchased from TaKaRa; Endotoxin-free plasmid extraction kit was purchased from QIAGEN; DMEM medium, fetal bovine serum were purchased from Gibco; Lipofectamine TM 2000 was purchased from Invitrogen;SsoFastTM EvaGreen Supermix was purchased from BIO-RAD, TFAP2c primary antibody, HIF-1α, anti-β-action antibody were purchased from Bioworld;EZ DNA Methylation-Gold Kit, methylation positive control and a negative control were purchased from ZYMO;HepG2 and H9C2 cell were conserved in our laboratory. ## Detection of mutations in cited2 gene Blood DNA was extracted from the CHD and healthy children. CITED2 coding region was cut into S1 and S2 to design primers. The primer sequences used in the assays are shown in [fig_ref] Table 2: Specific primers designed for the experiments [/fig_ref]. PCR amplification, and then sequencing were performed. DNA sequence was compared to the normal CITED2 coding sequence in the Genebank to reveal the mutation sites and types. Construct CITED2 mutant and wild-type recombinant plasmid CITED2 (c.573-578del6) and normal PCR purification products were connected with pMD19-T simple plasmid. Then they were transformed into competent E. coli DH5α and the positive clones were detected. Again, the positive clones were selected and sequenced. ## Transfection of plasmids into the cells Human hepatoma cell line HepG2 and Rats myocardial cell lines H9C2 were cultured in DMEM medium containing 10% fetal bovine serum. The medium was changed regularly. Recombinant plasmids were transfected into HepG2 and H9C2 using Lipofectamine™ 2000. The transfection efficiency of pEGFP-C1-wtCITED2 and pEGFP-C1-mtCITED2 were observed by fluorescence microscopy. The expression of CITED2 was analyzed with Western blotting. Experimental groups included: Empty vector group, Untransfected group, Mutation group and Wildtype control group. ## Detection of tfap2c and hif-1α mrna expression by q-pcr After transfection, the cells were cultured in the different conditions (21% O 2 , 74% N 2 , 5% CO 2 at 37°C or 1% O 2 , 94% N 2 , 5% CO 2 at 37°C). RNA was extracted after 24 h and reversely transcribed into cDNA. HIF-1α and TFAP2c was amplified by SsoFast™ EvaGreen Supermix. The sequences of the primers used in the assays are shown in [fig_ref] Table 2: Specific primers designed for the experiments [/fig_ref]. Expression differences of TFAP2c and HIF-1α mRNA was compared among the four assay groups. Each experiment was repeated at least three times. ## Detection of tfap2c and hif-1α protein levels by western blotting After transfecting, cells were respectively cultured in the condition of 37°C, 21% O 2 , 74% N 2 , 5% CO 2 and 37°C, 1% O 2 , 94% N 2 , 5% CO 2 . Proteins were collected after 48 h and boiled with buffer for 5 min and then separated by SDS-PAGE gel electrophoresis. Transferring protein onto PVDF and blocking 1 h with 5% skim milk. The membranes was incubated at 4°C overnight after hatching TFAP2c and HIF-1α antibodies. The PVDF was washed three times in PBST. TFAP2c and HIF-1α protein levels were compared among the four different groups. At least three independent experiments were performed. ## Detection of methylation in cited2 gene promoter region (cpg island) Genomic DNA was extracted from myocardial tissues of CHD and control groups and treated with bisulfite modification using an EZ DNA Methylation-Gold Kit. This treatment converts unmethylated cytosines to uracils, while methylated cytosines unchanged. CITED2 (−10 bp~−360 bp) region was detected by Bisulfite-PCR sequencing (BSP). The methylation sites were identified after PCR amplification of the cloning and sequencing. The methylation on CITED2 (−971 bp~−1171 bp) was detected using a methylation-specific PCR (MSP) assay. The methylated or unmethylated samples was analyzed by 2% agarose gel electrophoresis. ## Detection of cited2 mrna expression by q-pcr RNA was extracted from the methylated and control groups and reversely transcribed into cDNA. The transcriptional expression of CITED2 gene was detected and compared. β-actin was used as an internal control. CITED2: Sense: 5'-TTCCCTCACTTTCTCCAGTGCT CA-3 antisense: 5'-ATGAAGCGAGATGGCAGTTTGT GC-3', Length:191 bp; β-actin: Sense: 5'-CATGGGTCAG AAGGATTCCTATGTG-3' antisense: 5'-ATTTTCTCCAT GTCGTCCCAGTTG-3', length:116 bp. # Statistical analysis Statistical analysis was performed using student's t-test or Fish's exact test and X 2 test. The obtained P values # Results Mutations detected in CITED2 coding region 4 heterozygous mutations were detected among 120 patients with congenital heart disease, Including 2 cases of point mutations and 2 cases of deletion mutants. These mutations lead to corresponding amino acid variations in CITED2 protein. However no mutation were detected in samples from the 100 normal controls [fig_ref] Table 3: CITED2 gene mutations detected in pediatric patients with CHD [/fig_ref]. ## Measurement of tfap2c and hif-1α mrna expressions CITED2 wild-type and mutant recombinant plasmids were constructed successfully. By comparison to the wild-type plasmid pEGFP-C1-wtCITED2, the recombinant mutant plasmid pEGFP-C1-mtCITED2 could be identified by the absence of six bases based on sequencing. TFAP2c mRNA expression levels were reduced significantly in mutant group comparing to the wildtype group in H9C2 (P < 0.05). The empty vector group and untransfected group showed no statistical difference (P > 0.05). By contrast, HIF-1α mRNA expression levels were higher in the mutant group than that in wild-type group (P < 0.05) [fig_ref] Figure 1: Expression of TFAP2c, HIF-1α mRNA and protein after the transfection of CITED2... [/fig_ref]. Similarly, in HepG2 cells, we aslo found the same results, TFAP2c mRNA expression levels were reduced in mutant group comparing to the wild-type group, But HIF-1α mRNA expression levels were higher in the mutant group than that in wild-type group (P < 0.05) [fig_ref] Figure 1: Expression of TFAP2c, HIF-1α mRNA and protein after the transfection of CITED2... [/fig_ref]. ## Determination of protein expression levels of tfap2c and hif-1α The concentrations of TFAP2c protein were reduced in the mutant group comparing to that of the empty vector group in H9C2 (P < 0.05). TFAP2c protein concentrations were significantly lower in the mutant group comparing to the wild-type control group (P < 0.05). The empty vector group and untransfected group showed no significant difference (P > 0.05). Similarly to the mRNA data, the concentrations of HIF-1α protein was higher in the mutant group than that of the wild-type control group (P < 0.05) [fig_ref] Figure 1: Expression of TFAP2c, HIF-1α mRNA and protein after the transfection of CITED2... [/fig_ref]. Similarly, in HepG2 cells, It shows the same results, TFAP2c protein concentrations were lower in the mutant group comparing to the wildtype control group (P < 0.05), but the concentrations of HIF-1α protein was higher in the mutant group than that of the wild-type control group (P < 0.05) [fig_ref] Figure 1: Expression of TFAP2c, HIF-1α mRNA and protein after the transfection of CITED2... [/fig_ref]. ## Detection of methylation in cited2 promoter region Two methods were used to detect the methylation in gene promoter region. One was to use bisulfate-PCR primers (BSP) and the other was to use methylationspecific PCR primers (MSP). Both primers were described in Materials and Methods. The sequencing data from BSP showed that methylation was detected in 10 cases out of 12 patient samples tested (83% of methylation positive rate). The samples from the normal control group were small with only two cases. However, no methylation was detected in the control samples [fig_ref] Figure 2: The clone sequencing and Electrophoresis by BSP and MSP [/fig_ref]. The MSP showed that the methylation was detected in 16 cases out of 19 patients with CHD (84% of methylation-positive rate). No methylation was detected using this method in 2 normal control samples [fig_ref] Figure 2: The clone sequencing and Electrophoresis by BSP and MSP [/fig_ref]. It was also found that CITED2 expression levels were significantly decreased in samples with methylation in CITED2 promoter region compared to that of the samples without methylation in CITED2 (P < 0.05) [fig_ref] Figure 3: Expression of CITED2 mRNA in CITED2 methylated group and control group [/fig_ref]. # Discussion CITED2 gene is one important member of the CITED family. It is located in chromosome 6q23.3 and contains three exons and two introns, encoding a protein with 270 amino acids. The amino acid sequence of CITED2 protein is characterized by three conserved regions (CR1, CR2 and CR3). CR2 is a conserved region consisting of 32 amino acids, which binds with CH1 domain of CBP/P300. A unique serine-glycine-rich region (SGJ) is located in its carboxyl terminus. CITED2 gene has about 3 kb of CpG islands in its promoter region and coding region that may be modified by methylation [bib_ref] Molecular cloning and chromosomal localization ofthe human CITED2 gene encoding p35srj/Mrg1, Leung [/bib_ref]. A: Q-PCR analysis showed TFAP2c mRNA expression levels were reduced significantly in mutant group comparing to the wild-type group in H9C2 (*P < 0.05). By contrast, HIF-1α mRNA expression levels were higher in the mutant group than that in wild-type group (*P < 0.05). B: Q-PCR analysis showed TFAP2c mRNA expression levels were reduced significantly in mutant group comparing to the wild-type group in HepG2 (*P < 0.05). By contrast, HIF-1α mRNA expression levels were higher in the mutant group than that in wild-type group (*P < 0.05). C: Western-blotting data demonstrated that TFAP2c protein concentrations were significantly lower in the mutant group comparing to the wild-type control group in H9C2 (*P < 0.05). But the concentrations of HIF-1α protein was higher in the mutant group than that of the wild-type control group (*P < 0.05). D: Western-blotting data demonstrated that TFAP2c protein concentrations were significantly lower in the mutant group comparing to the wild-type control group in HepG2 (*P < 0.05). But the concentrations of HIF-1α protein was higher in the mutant group than that of the wild-type control group (*P < 0.05). in embryos can cause an abnormal heart ring formation, as well as various cardiac malformations including artial and ventricular septal defects, transposition of great arteries, double outlet right ventricle, tetralogy of fallot and so on [bib_ref] Epiblastic Cited2 deficiency resultsin cardiac phenotypic heterogeneity and provides a mechanism for..., Macdonald [/bib_ref]. Heart-specific knockout of CITED2 in mice leads to a ventricular septal defect and ventricular wall thinning, as well as abnormal angiogenesis, suggesting that CITED2 plays a vital role in the growth and development of ventricular muscles and ventricular septal coronary vessels [bib_ref] A cell-autonomous role of Cited2 in controlling myocardial andcoronary vascular development, Simon [/bib_ref]. CITED2 can act as a transcription cofactor assisting various transcription factors to modulate a normal development of the heart. CITED2−/− embryos can down-regulate Nodal, Lefty2 and Pitx2 expression in the left side plate mesoderm, which hampers the formation of the normal axis of the mouse heart [bib_ref] Cited2is required both for heart morphogenesis and Establishment of the left-right axis..., Weninger [/bib_ref]. CITED2 and HIF-1α can competitively bind on CH1 domain of CBP/P300. So CITED2 has been proposed to be a negative-feedbacked inhibitor under a hypoxic environment to limit HIF-1α overexpression. This negative feedback action is diminished when CITED2 gene is deleted or downregulated, which leads to an overexpression of HIF-1α [bib_ref] Identification of CITED2 as a negative regulator of fracture healing, Lee [/bib_ref] [bib_ref] Cited2 Modulates hypoxia-inducible factor-dependent expression of vascular endothelial growth factor in nucleus..., Agrawal [/bib_ref] [bib_ref] Hif1a down-regulation is associated with transposition of great arteries in mice treated..., Amati [/bib_ref]. Our study has further confirmed that CITED2 can, indeed, inhibit HIF-1α expression since this inhibition is diminished by CITED2 mutations detected in pediatric patients with CHD. As a consequence, the HIF-1α expression is enhanced in pediatric CHD patients with CITED2 mutations. The overexpression of HIF-1α might bring the following events that are associated with the development of CHD: 1) HIF-1α can suppress cell apoptosis in cardiac outflow tract. Cell apoptosis will be inhibited because of HIF-1α overexpression, which hinders the development of cardiac outflow tract. 2) HIF-1α overexpression can increase the expression of its target gene VEGF, which blocks the development of vasculature and leads to myocardial ischemia and hypoxia. 3) HIF-1α overexpression inhibits migration and transition of neural crest cell, which hinders the formation of a normal heart. The CR3 binding domain of CITED2 can bind with TFAP2, and its CR2 binding domain can bind CBP/ P300, So CITED2 as a bridge between TFAP2 and CBP/ P300 can activate the transcription activity of TFAP2. Lower TFAP2 transcriptional activity in CITED2−/− mice confirmed this view [bib_ref] Physical and functional interactions among AP-2 transcription factors, p300/CREB-binding protein, and CITED2, Braganca [/bib_ref]. TFAP2c and TFAP2a can interact with CITED2, especially TFAP2c [bib_ref] Redundant activities of Tfap2a and Tfap2c are required for neural crest induction..., Li [/bib_ref]. In our study the connection between TFAP2c and CBP/P300 weaked owing to CITED2 mutation, so there is a reduction in TFAP2c. Abnomal expression of TFAP2c can limit the development of cardiac neural rest. TFAP2c low expression further affect transcriptional regulator Pitx2 expression, which hamper the formation of the normal axis of the heart. Methylation is a process that transfers -CH3 to the specific base. Gene methylation plays a critical role in cell proliferation, differentiation and apoptosis. Aberrant methylation was mainly induced by environmental factors. And the incidence of congenital heart disease is closely associated with environmental factors during the pregnancy. Various factors during the pregnancy, such as ethanol, riboflavin and folic acid, can induce gene methylations in vivo [bib_ref] Alteration of gene expression by alcohol exposure at early neurulation, Zhou [/bib_ref] [bib_ref] The impact of recent alcohol use on genome wide DNA Methylation signatures, Philibert [/bib_ref] [bib_ref] Foetal nicotine exposure causes PKC1 gene repression by promoter methylation in rat..., Lawrence [/bib_ref] [bib_ref] Low dietary choline and low dietary riboflavin during pregnancy influence reproductive outcomes..., Chan [/bib_ref] [bib_ref] Feeding a proteinrestricted diet during pregnan inducesaltered epigenetic regulation of peroxisomal proliferator-activated..., Slater-Jefferies [/bib_ref]. Studies have shown that maternal high-fat diet combined with CITED2 gene deletion can increase penetrance of the heart abnormality body axis [bib_ref] Maternal high-fat diet interacts with embryonic Cited2 genotype to reduce Pitx2c expression..., Bentham [/bib_ref]. A close correlation has been discovered between congenital heart disease with the CITED2 gene deletion and environmental alteration. Zhu et al. reported that several abnormally methylated bases were found in ventricular septal defects and NOX5 hypermethylation in CpG island can inhibit its expression. Our study confirms that methylation of CITED2 gene promoter region is observed in pediatric patients with CHD and the methylation can decrease CITED2 transcription activity. # Conclusions In conclusion, our study discovers the CITED2 gene mutations as well as CITED2 gene promoter region methylation in pediatric patients with CHD, which affects transcriptional activity of TFAP2c and HIF-1α. The latter are closely associated with the heart development. Our data suggest that both gene mutations and epigenetic modifications play a role in the development of CHD. ## Consent Written informed consent was obtained from the patient for the publication of this report and any accompanying images. Expression of CITED2 mRNA in CITED2 methylated group and control group. It shows that CITED2 expression levels were significantly decreased in samples with methylation in CITED2 promoter region compared to that of the samples without methylation in CITED2 gene (*P < 0.05). [fig] Figure 1: Expression of TFAP2c, HIF-1α mRNA and protein after the transfection of CITED2 wild-type and mutant recombinant plasmids. [/fig] [fig] Figure 2: The clone sequencing and Electrophoresis by BSP and MSP. A. The clone sequencing of reverse complementary chain in CHD and control group by BSP. a: It shows the methylated sequence in CHD group, arrow represent methylated sites; b: The corresponding non-methylated sequences in control group, arrow represent unmethylated sites. B. It shows the result of Electrophoresis by MSP. it demonstrates that Methylated strip exsits in the samples with CHD,but in the control group it shows the Unmethylated strip. It demonstrates that CITED2 methylation exsits in CHD group. 1: DNA Marker 100-600 bp, 2: Blank control (ddH2O), 3: Methylated positive control, 11: Negative control, 4-8: Methylated samples in congenital heart disease, 9-10: Unmethylated samples in the control group, M: methylated strip, U: unmethylated strip. [/fig] [fig] Figure 3: Expression of CITED2 mRNA in CITED2 methylated group and control group. It shows that CITED2 expression levels were significantly decreased in samples with methylation in CITED2 promoter region compared to that of the samples without methylation in CITED2 gene (*P < 0.05). [/fig] [table] Table 1: Phenotypies and clinical manifestations of CHD in pediatric patients [/table] [table] Table 2: Specific primers designed for the experiments [/table] [table] Table 3: CITED2 gene mutations detected in pediatric patients with CHD [/table]

Long-term growth of temperate broadleaved forests no longer benefits soil C accumulation It is widely recognized that the long-term growth of forests benefits biomass carbon (C) sequestration, but it is not known whether the long-term growth of forests would also benefit soil C sequestration. We selected 79 representative soil profiles and investigated the influence of the forest stand age on the soil C dynamics of three soil layers (0-10, 10-20 and 20-30 cm) in temperate broadleaved forests in East China. The results suggest that the soil C density in temperature broadleaved forests significantly changes with the stand age, following a convex parabolic curve. At an early stand age, the soil C density usually increases, reaching its peak value at a pre-mature stand age (approximately 50 years old). At later stand ages, the soil C density usually decreases. Therefore, our results reveal a turning point in the soil C density at a pre-mature stand age. The long-term growth of temperate broadleaved forests after pre-mature stand age no longer benefits soil C accumulation, probably promotes topsoil C loss. In addition, we found that the soil C density in the upper soil layer usually changes with the forest stand development more significantly than that in deeper soil layers. Soil carbon (C) in forests has attracted much attention in recent years because its stability contributes to the mitigation of climate change [bib_ref] A review of research progress and future prospective of forest soil carbon..., Liu [/bib_ref] [bib_ref] Soil carbon stocks and forest biomass following conversion of pasture to broadleaf..., Cook [/bib_ref]. It was discovered that the soil C pool in temperate forests appears to be stable under disturbances, such as logging, wind storms, and invasive species [bib_ref] Carbon storage along a stand development sequence in a, Davis [/bib_ref]. It is widely recognized that the soil C pool in forests varies dynamically with the stand age [bib_ref] Estimating soil carbon reservoir of terrestrial ecosystem in China, Wang [/bib_ref] [bib_ref] Distribution of soil inorganic carbon storage and its changes due to agricultural..., Wu [/bib_ref] , but there are disagreements on how this occurs, leaving forest managers with uncertainty on how to best update forests for optimal C sequestration in the soil. [bib_ref] Loss and recovery of ecosystem carbon pools following stand-replacing wildfire in Michigan..., Rothstein [/bib_ref] observed a weak decline in the surface soil C content with the stand age in Michigan jack pine forests [bib_ref] Loss and recovery of ecosystem carbon pools following stand-replacing wildfire in Michigan..., Rothstein [/bib_ref]. On the contrary,discovered that the soil C increased by 1.1 Mg ha −1 yr −1 (1 Mg = 10 6 g) over the stand age range of 4~20 years in secondary tropical forests in Costa Rica. [bib_ref] Carbon storage in a chronosequence of Chinese fir plantations in southern China, Chen [/bib_ref] discovered that the soil C pool in a Chinese fir plantation declined at young stand ages and then re-accumulated C at the stand ages of 16 ~ 21 years 9 . [bib_ref] Soil carbon change and its affecting factors following afforestation in China, Shi [/bib_ref] argued, by summarizing 70 publications, that the highest soil C accumulation rate occurred at stand ages of 10-20 years old [bib_ref] Soil carbon change and its affecting factors following afforestation in China, Shi [/bib_ref]. By summarizing more than 100 publications, [bib_ref] Carbon and nitrogen dynamics during forest stand development: a global synthesis, Yang [/bib_ref] argued that the soil C pool did not undergo significant changes during forest stand development in most studies [bib_ref] Carbon and nitrogen dynamics during forest stand development: a global synthesis, Yang [/bib_ref]. Therefore, it remains uncertain how the soil C changes in the long-term growth process of forests. In China, secondary forests have expanded due to reforestation over the past half century. Seven national-scale forest investigations have been performed since the 1950 s, but focused only on the timber volume and forest C biomass, without considering the soil C 12 . National-scale soil investigations have also been performed, but unfortunately, they focused on the soil C in different soil types rather than for vegetation types [bib_ref] Regional patterns of soil organic carbon stocks in China, Yu [/bib_ref] [bib_ref] National scale analysis of soil organic carbon storage in China based on..., Yu [/bib_ref]. Some studies estimated the soil C pool in Chinese forests based on process-based BIOME models 15 , but could not resolve the complicated relationship between the soil C and forest stand age. Thus, it is necessary to elucidate how the soil C changes with the forest stand age to provide scientific evidence for forest management. In this paper, we investigated 79 representative soil profiles in temperate broadleaved forests in eastern China. The objectives were to uncover the relationship between the soil C sequestration and the stand age of temperate broadleaved forests to improve forest management. # Results Change trend of soil C density with stand age. Regardless of the broadleaved tree species, we use the actual forest stand age as the independent variable to examine how the soil C density changes in three soil layers (0-10, 10-20, 20-30 cm) in temperate broadleaved forests. When the three soil layers were taken as a unit, the results show that the soil C density is significantly correlated with the forest stand age. The soil C density changes with the stand age following a convex parabolic curve (R 2 = 0.3273), not a straight line. The soil C density increases at a young stand age, reaches its maximum carbon storage at an average age of approximately 50 years, and then gradually declines with the increasing stand age [fig_ref] Figure 1: Soil C density change with actual stand age in soil layers [/fig_ref]. Therefore, the results indicate that there exists a turning point of soil C density in temperate broadleaved forests during stand age development. The soil acts as a C sink following forest establishment, but switches to a C source at approximately 50 years old, implying that the long-term growth of temperate broadleaved forests after 50 years no longer benefits soil C accumulation, but rather contributes to C loss from the soil. When comparing the three soil layers, a significant change in the soil C density with the forest stand age following a parabolic curve was observed (R 2 = 0.4309) in the upper soil layer (0-10 cm). In the soil layer of 10-20 cm, the soil C density also varied in a parabolic curve with the forest stand age (R 2 = 0.2346), but the peak of the parabolic curve became lower. In the deeper soil layer of 20-30 cm, the peak of the parabolic curve disappeared (R 2 = 0.0193), as the soil C density changed only slightly compared to in the upper soil layers [fig_ref] Figure 2: Soil C density change with actual stand age in three soil layers [/fig_ref]. As a result, the soil C in the upper layers is more sensitive to the forest stand age than that in the lower soil layers. Average change rate of soil C density with stand age class. To quantify the soil C dynamics with the stand age class, we divided the entire growth sequence of temperate broadleaved forests into five stand age classes (young, middle, pre-mature, mature and over-mature) . When the three soil layers (0-10, 10-20, 20-30 cm) were taken into account as a whole, the results suggest that the soil C density reaches its peak value (approximately 85.6 Mg C/ha) at the pre-mature stand age. On average, the soil C density increased at a rate of 0.813 Mg C/ha per year prior to the pre-mature stand age. Subsequently, it declined at a rate of 0.74 Mg C/ha per year after the pre-mature stand age and to 56.0 Mg C/ha at the over-mature stand age (average 91 years old). Therefore, the quantitative results indicate that the pre-mature stand age (average 52 years old) is a turning point in the soil C dynamics. The soil C accumulated at a rate of 0.813 Mg C/ha per year before the turning point and then exhibited a loss of 0.74 Mg C/ha per year after the turning point [fig_ref] Figure 3: Soil C dynamics with stand age class in three soil layers [/fig_ref]. Comparing the three soil layers, the upper soil layer (0-10 cm) showed the most significant change in soil C density, with an increase of 0.643 Mg C/ha per year from young to pre-mature and a decrease of 0.398 Mg C/ha per year from pre-mature to over-mature. The middle soil layer (10-20 cm) showed an increase of 0.268 Mg C/ha per year from young to pre-mature and a decrease of 0.253 Mg C/ha per year from pre-mature to over-mature. The deepest soil layer (20-30 cm) showed a slightly fluctuating soil C density without any significant change over the entire growth sequence. # Discussion Our results demonstrate that the soil C density in temperate broadleaved forest changes with the stand age following a convex parabolic curve, and there exists a turning point with a single peak in the soil C accumulation at approximately 50 years old (Figs 1, 2 and 3). However, the results only show a general (significant single-peak curve) change trend of soil C density, but omits minor changes along the successional process since the data is not enough to identify the minor changes. The soil C density probably changes following a multi-peak curve with no more than one turning point during the entire forest stand age, as some studies reported that the soil C initially decreased or increased slowly for the first decade after afforestation and then began to accumulate quickly with the stand age. The multi-peak curve of soil C density usually exist under the precondition of afforestation. For example,synthesised available world-wide information on changes in soil C after afforestation, and argued that soil C in surface soil (< 10 or < 30 cm depth) initially declines during the first 5 years after establishing a plantation but recovers by the age of 30 years. The initial decline of soil C incame from the average data in the 43 published or unpublished studies, so the decline is not significant since the data are highly variable, with soil C either increasing or decreasing in young (< 10 year) forest stands. [bib_ref] Biomass and carbon storage in an age-sequence of Korean Pine (Pinuskoraiensis) plantation..., Li [/bib_ref] observed the total mineral soil C initially appeared to decline at the early stand age, but recovered by the stand age of 35 years for coniferous plantation forest with Korean Pine (Pinus koraiensis). It is a pity that the natural change of soil C is not credible after 35-year-old stand because the soil C suffered from disturbance greatly from human, such as thinning treatment to the35-and 51-year-old stands [bib_ref] Biomass and carbon storage in an age-sequence of Korean Pine (Pinuskoraiensis) plantation..., Li [/bib_ref]. [bib_ref] Afforestation with Norway spruce on a subalpine pasture alters carbon dynamics but..., Hiltbrunner [/bib_ref] examined the effects of afforestation with Norway spruce (Picea abies L.) in a grazed subalpine pasture in Switzerland on soil organic carbon (SOC), and discovered that soil C stock decreased after tree establishment, reaching a minimum 40-45 years after afforestation, and increased thereafter [bib_ref] Afforestation with Norway spruce on a subalpine pasture alters carbon dynamics but..., Hiltbrunner [/bib_ref] at the 0-30 cm soil layer following afforestation in Northern Europe by a meta-analysis, revealed that SOC loss generally for barren, cropland, heathland and grass-land at the initial phase following afforestation (0-30 years). The detectable gains in SOC stocks appear in later stages (> 30 years), especially for afforestation of croplands [bib_ref] Soil carbon stock change following afforestation in Northern Europe: a meta-analysis, Barcena [/bib_ref]. Yu et al. [bib_ref] Soil carbon budget in different-aged Chinese fir plantations in south China, Yu [/bib_ref] investigated the soil C in four Chinese fir (Cunninghamia lanceolata Hook) plantations (Chinese fir was planted in clear-cut sites in natural broad-leaved forest) in Jiangxi Province, south China, discovered that soil C density at the depth of 0-20 cm declined before 16 years, but increased after 16 years, since soil C density declined from 35.98 Mg·ha −1 in the 7-year plantation to 30.12 Mg·ha −1 in the 16-year plantation, and then increased after 16 years old [bib_ref] Soil carbon budget in different-aged Chinese fir plantations in south China, Yu [/bib_ref]. Therefore, we can speculate that soil C density probably changes following a multi-peak curve, and another turning point of the soil C density may exist in the early decades of afforestation, besides the large turning point at approximately 50 years old [fig_ref] Figure 4: Multi-peak curve of topsoil C density change with forest stand age according... [/fig_ref]. In the later stage of forest development, many reports indicate that old forests are expected to maintain their biomass accumulation for a long time through the development of a multilayer canopy structure 21 , but this does not mean that the soil C can continue to increase as long as the biomass accumulates. Our results show that old-growth forest could not sustain the soil C increase due to the decreasing soil C density after the pre-mature stand age (average stand age 50 years old). However, there seems to be some controversy on this point. It is conventionally accepted that the soil C levels in old-growth forests are in a steady state [bib_ref] Old-growth forests as global carbon sinks, Luyssaert [/bib_ref] [bib_ref] Carbon pools in a montane old-growth Norway spruce ecosystem in Bohemian Forest:..., Seedre [/bib_ref]. However, [bib_ref] Old-growth forests can accumulate carbon in soils, Zhou [/bib_ref] reported that soils in the top 20-cm soil layer in preserved old-growth forests (age > 400 years) in southern China accumulated C significantly at an unexpectedly high rate from 1979 to 2003 23 . Li & Liu (2014) argued that an old forest (38-56 y) was able to continuously accumulate C in the soil in China's Loess Plateau, even when the biomass significantly decreased [bib_ref] Age-related Changes of Carbon Accumulation and Allocation in Plants and Soil of..., Li [/bib_ref]. These different opinions could be partly attributed to the different definitions of "old forest" with tree species and environment spatial variability, as there is currently no recognized definition. For example, a stand age of 38-56 years old in the study of [bib_ref] Age-related Changes of Carbon Accumulation and Allocation in Plants and Soil of..., Li [/bib_ref] was regarded as old forest [bib_ref] Age-related Changes of Carbon Accumulation and Allocation in Plants and Soil of..., Li [/bib_ref] , equivalent to the pre-mature stands age (40-60 years old) in our study. It is likely that tree species and environment spatial variability are the leading causes of the different opinions, as different environments and tree species can affect the carbon accumulation-and-release processes [bib_ref] Carbon and nitrogen dynamics during forest stand development: a global synthesis, Yang [/bib_ref] [bib_ref] Biogeochemical and hydrological controls on carbon export from a forested catchment in..., Kawasaki [/bib_ref] [bib_ref] Variation in carbon storage and its distribution by stand age and forest..., Wei [/bib_ref] [bib_ref] Sources of errors and uncertainties in the assessment of forest soil carbon..., Vanguelova [/bib_ref]. The synergistic effects of many factors should be further explored to uncover the complex mechanism of soil C dynamics [bib_ref] Distinct temperature sensitivity of soil carbon decomposition in forest organic layer and..., Xu [/bib_ref] [bib_ref] Maximum temperature accounts for annual soil CO 2 efflux in temperate forests..., Zhou [/bib_ref] [bib_ref] Factors controlling soil organic carbon stability along a temperate forest altitudinal gradient, Tian [/bib_ref] [bib_ref] Differential controls on soil carbon density and mineralization among contrasting forest types..., You [/bib_ref]. # Conclusions Our results show that soil C in temperature broadleaved forests significantly changes with stand age. Generally, it exhibits a change trend in the shape of a convex parabolic curve with stand age, regardless of the tree species. At the early stage of forest development, the soil C density usually increases, and it reaches its peak value at the pre-mature stand age (approximately 50 years old). At later stages of forest development, the soil C density usually decreases. This phenomenon provides strong evidence that there is a turning point of the soil C density in temperate broadleaved forests at the pre-mature stand age, when the soil switches from being a net C sink to a net C source. Therefore, we drew the conclusion that the long-term growth of temperate broadleaved forests after pre-mature stand age no longer benefits soil C accumulation. Our study also confirmed that the soil C in the upper layers is more sensitive to forest stand age than that of the lower soil layers, as the soil C density in the upper soil layers usually changes significantly with the forest stand development. # Materials and methods Study area. The study area covers approximately 139,000 km 2 in the Anhui Province (114°51′ -119°36′ E, 29°26′ -34°37′ N) of East China [fig_ref] Figure 5: Location of study area and sampling plots of 79 representative soil profiles... [/fig_ref]. The Asian monsoon circulation creates a temperate continental monsoon climate with an annual average temperature of 14-17 °C and an annual precipitation of 800-1800 mm. The soil type is yellow brown soil 32 . Three mountain ranges (Dabie, Jiuhua and Huang Mountains) lie in the southwestern and southern regions [fig_ref] Figure 5: Location of study area and sampling plots of 79 representative soil profiles... [/fig_ref] and are covered with temperate and subtropical forests. The dominant forest types are temperate deciduous broadleaved forests, coniferous forests, and mixed forests. In recent decades, large-scale deforestation has been curbed, and reforestation projects have been carried out, providing an opportunity to resume normal forest development [bib_ref] Changes in forest biomass carbon storage in China between, Fang [/bib_ref]. Forest managers also allow people to update some forests to obtain timber for money. Therefore, the study area contains various forests with stand ages ranging from 0 to 100 years. However, it is unclear what updating schedule for the forest is the most favourable for soil C sequestration. ## Sampling sites (plots). To avoid the effect of the spatial heterogeneity of sampling sites on SOC, we did our utmost to select coincident sampling sites (plots) in vegetation composition, soil type, and the same development process. All the sampling sites must have typical temperate broadleaved forest which is determined by climatic zones, though there are other forests, such as coniferous forests, conifer and broadleaf mixed forests. To avoid of human disturbance, all the sampling sites were selected in protecting natural forests to ensure a natural growth process. Young broadleaved forests being selected should have similar land use process because SOC in young forests suffer more effect from previous land use. We didn't consider the young forests which land use type had been changed greatly by human. Thus, almost all forest vegetation in sampling sites belongs to secondary successional vegetation under the protection of human beings. We selected the typical forests for every stand ages (young, middle, pre-mature, mature, over-mature) according to the natural succession of temperate broadleaved forest, so there is a slightly inconsistent in vegetation (species) composition for different stand age due to the natural succession. The vegetation composition for different stand ages is listed as follows [fig_ref] Table 2: The vegetation composition in sampling sites for different stand ages of temperate... [/fig_ref]. The soil in study area is classified as yellow brown soil zone according to "Map of Soil Regionalization of China" [bib_ref] The soil regions of China, Xi [/bib_ref]. The sampling sites in our study ensured a typical yellow brown soil, and other soil types were avoided. Generally, the sampling sites are coincident approximately in vegetation composition, soil type and development process, in spite of existing spatial heterogeneity more or less. ## Soil sampling. To examine the dynamics of the soil C with stand age, 79 soil profiles of sampling sites were investigated in representative temperate broadleaved forests in September of both 2011 and 2012 [fig_ref] Figure 5: Location of study area and sampling plots of 79 representative soil profiles... [/fig_ref]. The actual stand age of each forest type was recorded by visiting farmers and forest management staff. The investigation focused on soil carbon density in the 0-30 cm soil layer, since soil carbon in the layer accounts for the majority of the soil profile 0-100 cm, and is sensitive to forest stand ages more than that in deeper soil layers. Vertical soil profiles were dug in the sampling plots, and soil samples were collected from three soil layers (0-10, 10-20, 20-30 cm) using a ring knife (volume of 100 cm 3 ) for measuring the soil bulk density and a spade for measuring the soil C content. Soil samples from the surface soil layer (0-10 cm) included the forest floor (O horizons, i.e., the organic horizon), but surface litter was not included in the calculation of the soil C. In addition, the relevant environmental information for each sampling plot was recorded, such as the geographical location with latitude and longitude, forest type and stand age. Data analysis. The soil samples collected with the ring knife were used to measure the soil bulk density by the drying method in the laboratory. The soil samples collected with the spade were air-dried, ground using a mortar, and passed through a 2-mm sieve to remove all roots and stones. Finally, chemical analyses were performed to measure the soil C concentration by dry combustion using an elemental analyser (vario MACRO cube, Elementar, Germany). For these soil profiles, we calculated the soil C density (D) of the three soil layers of 0-10, 10-20 and 20-30 cm using Equation (1), [formula] ∑ = × × = D D H C ( ) ,(1)i n i i i 1 [/formula] where D is the soil C density (10 5 g C/ha.), D i is the bulk density (g/cm 3 ), H i is the soil depth (cm), C i is the soil C concentration (‰), and i represents the three soil layers. [fig] Figure 1: Soil C density change with actual stand age in soil layers (0-30 cm) in temperate broadleaved forests in Anhui Province, East China. [/fig] [fig] Figure 2: Soil C density change with actual stand age in three soil layers (0-10, 10-20, 20-30 cm) in temperate broadleaved forests in Anhui Province, East China. Scientific RepoRts | 7:42328 | DOI: 10.1038/srep42328 [/fig] [fig] Figure 3: Soil C dynamics with stand age class in three soil layers (0-10, 10-20, 20-30 cm) in temperate broadleaved forests. Scientific RepoRts | 7:42328 | DOI: 10.1038/srep42328 [/fig] [fig] Figure 4: Multi-peak curve of topsoil C density change with forest stand age according to our study and other studies. [/fig] [fig] Figure 5: Location of study area and sampling plots of 79 representative soil profiles in temperate broadleaved forests in Anhui Province, East China (The map was generated using ArcGIS for Desktop 10.2, http://www.esri.com/software/arcgis). Scientific RepoRts | 7:42328 | DOI: 10.1038/srep42328 [/fig] [table] Table 2: The vegetation composition in sampling sites for different stand ages of temperate broadleaved forests in Anhui Province. [/table]

Change in Proportional Protein Intake in a 10-Week Energy-Restricted Low- or High-Fat Diet, in Relation to Changes in Body Size and Metabolic Factors Objective: To investigate in a secondary analysis of a randomised trial the effects of a low-/ high-fat diet and reported change from baseline in energy% from protein (prot%), in relation to changes in body size and metabolic factors. Methods: Obese adults (n = 771) were randomised to a 600 kcal energy-deficient low-fat (20-25 fat%) or high-fat (40-45 fat%) diet over 10 weeks. Dietary intake data at baseline and during the intervention were available in 585 completers. We used linear regression to calculate the combined effects of randomised group and groups of prot% change (<-2 /-2 to 2/>2) on outcomes. Results: The low-fat group with # Introduction Life-style interventions for obesity treatment entail reducing energy intake or increasing energy expenditure. The importance of macronutrient composition of a weight-reducing diet has been widely investigated [bib_ref] Atkins and other low-carbohydrate diets: hoax or an effective tool for weight..., Astrup [/bib_ref] [bib_ref] High-protein low-carbohydrate diets: what is the rationale?, Busetto [/bib_ref]. A low-fat, energy-restricted diet has been recommended for weight loss [bib_ref] High-protein low-carbohydrate diets: what is the rationale?, Busetto [/bib_ref] , but this has been challenged by results in a number of controlled trials that have shown greater effects on weight loss by high-fat, low-carbohydrate diets, compared with diets low in fat [bib_ref] Systematic review of randomized controlled trials of lowcarbohydrate vs. low-fat/low-calorie diets in..., Hession [/bib_ref] [bib_ref] Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet, Shai [/bib_ref] [bib_ref] Comparison of the Atkins, Zone, Ornish, and LEARN diets for change in..., Gardner [/bib_ref]. Randomised controlled trials have also indicated positive effects on weight loss [bib_ref] Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet, Shai [/bib_ref] [bib_ref] Comparison of the Atkins, Zone, Ornish, and LEARN diets for change in..., Gardner [/bib_ref] [bib_ref] Effects of variation in protein and carbohydrate intake on body mass and..., Krieger [/bib_ref] [bib_ref] Randomized trial on protein vs carbohydrate in ad libitum fat reduced diet..., Skov [/bib_ref] and weight maintenance [bib_ref] Diets with high or low protein content and glycemic index for weight-loss..., Larsen [/bib_ref] by a high protein intake. However, a large trial for 2 years showed no differences in weight loss between four diets with different proportions of protein, fat and carbohydrates; rather it showed that adherence to any diet was highly predictive of weight loss [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref]. This study and other randomised trials did not investigate the combined effects of dietary fat and protein on weight loss, as high protein and high fat intake were clustered in the randomised groups [bib_ref] Systematic review of randomized controlled trials of lowcarbohydrate vs. low-fat/low-calorie diets in..., Hession [/bib_ref] [bib_ref] Diets with high or low protein content and glycemic index for weight-loss..., Larsen [/bib_ref] [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] [bib_ref] The effects of high protein diets on thermogenesis, satiety and weight loss:..., Halton [/bib_ref]. Many participants in dietary intervention trials do not comply with the prescribed diets [bib_ref] Atkins and other low-carbohydrate diets: hoax or an effective tool for weight..., Astrup [/bib_ref] [bib_ref] Diets with high or low protein content and glycemic index for weight-loss..., Larsen [/bib_ref] [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] , and the differences in actual diet may influence the outcome. Analysis of actual or reported dietary intake in interventions may thus add important information to results derived from intention-to-treat analyses. In the Nutrient-Gene Interactions in Human Obesity (NUGENOB) trial, we previously reported results for the effect of a 10-week energy-restricted low-fat versus high-fat diet on weight loss and metabolic factors in obese men and women [bib_ref] Astrup A: Randomized, multi-center trial of two hypo-energetic diets in obese subjects:..., Petersen [/bib_ref]. Using the intention-to-treat approach in the analyses, we found no differences in weight loss or other measures of body size (waist, fat mass and fat-free mass) between the two diets, but participants on the low-fat diet had a greater reduction in total cholesterol concentration. The opposite was observed for plasma triglycerides. The trial includes individual data on dietary intake from weighed food records over several days before, during and at the end of intervention, which enables complementary analysis to the intention-to-treat approach. The aim of the present study was, in a re-analysis of the NUGENOB trial, to analyse the relation between randomised groups and recorded individual dietary intake and changes in body size and metabolic factors, with particular emphasis on the combination of dietary fat and protein. # Participants and methods ## Nugenob The NUGENOB trial ( www.nugenob.com ) has been previously described in detail [bib_ref] Astrup A: Randomized, multi-center trial of two hypo-energetic diets in obese subjects:..., Petersen [/bib_ref]. Briefly, 771 obese men and women were randomly assigned to a diet low or high in fat for 10 weeks. Participants were recruited to one of eight centres in seven European countries. Inclusion criteria were a BMI of at least 30 kg/m 2 and age 20-50 years. Participants were not included if they had experienced a weight change more than 3 kg within 3 months prior to the study or if they reported other pre-identified characteristics that could influence the results, such as pregnancy and medications or certain diseases. Written informed consent was retrieved from all study participants, and the project was approved by the ethical committee at each study centre. ## Dietary intervention The dietary targets for the two randomised groups were an intake of 20-25% of energy derived from fat (fat%) in the low-fat group and 40-45 fat% in the high-fat group. Both groups were prescribed a protein intake of 15 energy% (15 prot%) and no or minimal alcohol consumption. The goal for energy intake in the two groups was a daily energy deficit of approximately 600 kcal, calculated by (1.3 (resting energy expenditure (REE) in kcal/day) -600. Before the intervention, dieticians gave individual dietary instructions to participants at their study centre, and written dietary guidelines and examples of 24-hour menus adapted to the local customs were provided. Participants were asked to maintain their usual physical activity during the intervention. Participants completed weighed food recordings before the intervention and at week 2, 5 and 10. Recordings at baseline and week 10 included one weekend day and two weekdays, and recordings at week 2 and 5 included one weekday. The reported dietary intake was coded and analysed locally using countryspecific food databases. ## Anthropometry and biochemical analyses Participants underwent a clinical examination at their clinical centre. They were instructed to avoid strenuous exercise and abstain from drinking alcohol 3 days prior to the examination and to fast overnight and void the bladder before measurements. Height was measured with a calibrated stadiometer, and weight was measured in light indoor clothes without shoes on a calibrated scale. Waist circumference was measured mid-way between the lowest rib and the iliac crest at the end of a normal expiration, with the measuring tape held close to the body. The mean of three estimates of height, weight and waist circumference was recorded for each variable. Fat mass and fat-free mass were measured by bioelectrical impedance (Bodystat ® ; Quadscan 4000, Isle of Man, British Isles, UK). After 15 min rest, a venous blood sample was drawn. Plasma glucose (ABX diagnostics, Montpellier, France), triglycerides (Sigma, St. Louis, MO, USA; ABX diagnostics), total cholesterol (ABX diagnostics), and high-density lipoprotein cholesterol (HDL) (Roche, Indianapolis, IN, USA) were measured on a COBAS MIRA automated spectro-photometric analyzer (Roche Diagnostica, Basel, Switzerland). Free fatty acids (NEFA C kit; Wako Chemicals, Neuss, Germany) were measured on a COBAS FARAH centrifugal spectro-photometer (Roche Diagnostica, Basel, Switzerland). Plasma insulin was measured with a double antibody radio-immunoassay (Insulin RIA 100; Kabi-Pharmacia, Uppsala, Sweden). Low-density lipoprotein cholesterol (LDL) level was calculated by Friedewald's formula [bib_ref] Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use..., Friedewald [/bib_ref] from concentrations of total cholesterol, HDL and triglycerides, and homeostatic model assessment of insulin resistance (HOMA-IR) was calculated from glucose and insulin concentrations . Measurements of anthropometrics and biochemical analyses were performed shortly before and after the intervention (REE only before in some participants). ## Data preparation All variables of diet, anthropometry and metabolic factors were checked for outliers, and extreme outliers, defined as values not connecting with the tail of the distribution, were recoded to missing. For each variable, the number of excluded values was less than 5 except for fat mass and fat-free mass for which values were also excluded if they were considered unrealistic in relation to total body weight. Thus, in addition to 1 extreme outlier for change in fat mass (-20.5 kg), and 4 for change in fat-free mass (increase >9 kg), another 14 sets of values for fat mass and fat-free mass were excluded because the sum of the two measures differed by more than 2 kg from total body weight. # Statistical analysis This analysis involved 585 completers for whom dietary intake data were available. There were no differences in baseline characteristics between completers and non-completers, as previously reported [bib_ref] Astrup A: Randomized, multi-center trial of two hypo-energetic diets in obese subjects:..., Petersen [/bib_ref]. Differences in levels of BMI and baseline energy intake between completers with versus without dietary data were tested by two-sample t-test. We analysed the randomised groups and changes from baseline in individual dietary intake (energy intake, fat% and prot%) in relation to changes in body size (weight, waist circumference, fat mass and fat-free mass) and metabolic factors (glucose, insulin, HOMA-IR, free fatty acids, triglycerides, total cholesterol and LDL/HDL ratio). Change in protein intake in g/kg baseline body weight in relation to outcomes was also analysed for comparison with results for prot% change. Changes in dietary intake were calculated as the difference between mean intake over 3 days at baseline and the intake over 5 days during the intervention with equal weight for intake at the first part versus end of intervention. Changes in body size and metabolic factors refer to the difference between levels at baseline and directly after the intervention. We used linear regression to calculate associations (denoted beta) between the randomised low-/high-fat diet or change in dietary intake (independent variable) and changes in body size and metabolic factors (dependent variable). All models were adjusted for the baseline body size or metabolic factor of interest, sex, age and centre. Analysis of high versus low-fat diet was additionally adjusted for dietary fat% at baseline. In a second model, we further adjusted for change in energy intake. Linearity of associations was tested by the use of likelihood-ratio tests, in which the linear model was compared with a model that additionally included a quadratic term of dietary change. For non-linear associations, we present results for dietary change in groups. Under-reporting of food intake is common in dietary self-reports [bib_ref] Using intake biomarkers to evaluate the extent of dietary misreporting in a..., Subar [/bib_ref] ; so we checked whether exclusions of participants who were considered having a low reported energy intake affected the results for associations between change in energy intake and changes in body size. Sensitivity analyses were performed with exclusions of participants with the 10% lowest ratio of reported energy intake / REE before and during the intervention. In 187 participants without data on REE after the intervention, we used REE calculated from the equation by the FAO/WHO/UNU [bib_ref] Report of a joint FAO/WHO/UNU Expert Consultation: World Health Organ Tech, Energy [/bib_ref]. We further analysed the randomised low-/high-fat diet combined with changed prot% intake. Changed intake reflects both habitual intake at baseline and actual intake during the intervention, during which all participants had been prescribed 15 energy% protein. We analysed changed prot% intake in three groups: <-2%, -2% to 2%, and >2%. These groups were chosen so to ensure nutritionally relevant differences between the groups and a satisfactory number of participants in each group. We tested for interactions between fat group and prot% change, in relation to outcomes, by likelihood ratio tests for models with and without interaction terms. The trend in association across protein groups was calculated by linear regression with protein groups as a continuous variable with values 0, 1 and 2. Statistical analyses were performed in Stata version 9.2, and two-sided p values lower than 0.05 were considered nominally statistically significant. # Results ## Study participants Out of 648 (84%) completers of the trial, 585 had complete dietary intake data. Completers with versus without dietary intake data did not differ significantly with respect to baseline BMI (p = 0.3), or baseline energy intake (p = 0.06 in women and 0.1 in men). ## Baseline characteristics and dietary intake Baseline characteristics and 10-week changes are reported in table 1 . The mean baseline energy intake of 2,049 kcal/day in women and 2,641 kcal/day in men declined during the intervention by on average 567 kcal/day and 751 kcal/day respectively, but there was large variation in the reported change for energy intake as well as for fat and protein intake (supplemental [fig_ref] Table 1: Mean [/fig_ref] , available at http://content.karger.com/ProdukteDB/produkte.asp?doi=351726 ). Energy intake remained stable between week 2 and 10 [fig_ref] Table 1: Mean [/fig_ref]. In the three groups for changed prot% intake (<-2, -2 to 2 and >2), the mean (SD) prot% change was -4.3 (1.9), 0.1 (1.2) and 4.5 (1.9) respectively. Changes in absolute intake of fat, carbohydrates and total energy intake decreased by increasing prot% group . [fig_ref] Table 3: Beta for the effect of dietary intake on change in body size... [/fig_ref] shows the association between the low-fat/high-fat groups and the changes in body size or metabolic factors as well as that between the dietary intake and the changes of the very same outcome parameters. The decline in energy intake was significantly related to changes of all body size measures and to decreased plasma concentrations of triglycerides and total cholesterol. Sensitivity analyses with exclusions of participants with a very low reported energy intake did not materially influence the associations between changed energy intake and anthropometric measures. ## Effects of changes in dietary intake The previously shown [bib_ref] Astrup A: Randomized, multi-center trial of two hypo-energetic diets in obese subjects:..., Petersen [/bib_ref] greater decline in triglyceride concentration in the high-fat diet (p = 0.004) and a greater decline of cholesterol in the low-fat diet (p = 0.003), but no other significant effects on outcomes, persisted after adjustment for baseline fat% intake and Fat%, Carb%, Prot% and Alc% = percent energy intake from fat, carbohydrates, protein and alcohol respectively. a Data were missing or extreme values were excluded for: fat mass, 20 participants; fat-free mass, 23; waist circumference, 4; glucose, 4; insulin, 11; HOMA-IR, 11; free fatty acids, 1; triglycerides, 6; cholesterol, 2; LDL, 10; HDL, 2; LDL/HDL ratio, b Change of measures of body size and metabolic factors refers to the difference between baseline and after the intervention. Change in dietary intake is the difference between reported intake at baseline (day 1 + day 2 + day 3) / 3 and at week 2 and/or 5, and week 10 ((week 2 + week 5) / 2) + ((week 10, day 1 + day 2 + day 3) / 3) / 2. change in energy intake. As expected, results for change in reported fat% intake pointed in the same direction as the results for randomised low-/high-fat diet. Increased prot% intake was related to decreased cholesterol concentration (p = 0.01), which diminished after adjustment for change in energy intake. An increased, or less decreased, absolute protein intake/kg baseline body weight resulted in less body size reduction (weight, p = 0.02; fat-free mass, p = 0.02; waist, p = 0.02), but the associations became non-significant after adjustment for changed energy intake. Protein change in g/kg weight showed a significant inverse association (p = 0.03) with change in cholesterol after adjustment for change in energy intake. ## Effects of low-fat or high-fat diet combined with change in prot% intake Increased prot% intake in the low-fat group was related to greater weight loss (p for trend = 0.05), but no such pattern was shown in the high-fat group [fig_ref] 2: Mean a [/fig_ref]. The low-fat group with a >2 unit prot% increase showed the greatest weight loss, and the poorest effect was shown in the contrasting high-fat group with >2 units decreased prot% intake (β = 1.10, p = 0.03). Cholesterol showed the same pattern. The difference in cholesterol reduction between these groups was 0.25 mmol/l (p = 0.003), which was almost double the difference between the low-fat and high-fat group not taking prot% change into account. Adjustments for changed energy intake somewhat weakened the associations. There was a significant interaction between low-fat/high-fat diet and groups of prot% change in relation to change in triglyceride concentration (p = 0.01) [fig_ref] Figure 3: Fig [/fig_ref]. In the low-fat group, an increasing prot% intake was related to reduced triglyceride concentration (p for trend < 0.001), but there was no such trend in the high-fat group. The difference of 200 μmol/l between the groups of >2 unit prot% increase and >2 prot% decrease in the low-fat diet was 2.5 times larger than the difference between the low-fat and high-fat diet. The effects on changes in weight and cholesterol by change in protein in grams showed an opposite trend to that in analyses of prot% combined with fat group (supplemental [fig_ref] 2: Mean a [/fig_ref] , available at http://content.karger.com/ProdukteDB/produkte.asp?doi=351726 . Note a different ordering of protein groups in [fig_ref] 2: Mean a [/fig_ref]. The correlation between change in protein intake as energy% and g/kg body weight, and the overlap between categories of the two measures, is shown in supplemental [fig_ref] Figure 3: Fig [/fig_ref] (available at http://content.karger. com/ProdukteDB/produkte.asp?doi=351726 ). # Discussion In this 10-week randomised trial of two energy-restricted diets low or high in fat, participants on the low-fat diet who modified their diet such that the proportion of remaining energy from protein was higher than the proportion in their usual diet (increase in prot% intake >2 units) showed the greatest weight loss and decline in plasma cholesterol. The lowest weight loss and cholesterol decline was shown in participants on the high-fat diet with a >2 unit decreased prot% intake. These differences for weight loss and cholesterol change were b Difference between reported intake at baseline (day 1 + day 2 + day 3) / 3 and at week 2 and/or 5, and week 10 ((week 2 + week 5) / 2) + ((week 10, day 1 + day 2 + day 3) / 3) / 2. c Difference between baseline and after the intervention. d Adjusted for the baseline body size or metabolic factor of interest, sex, baseline body size/metabolic factor × sex, age, and centre. High-fat versus low-fat diet was additionally adjusted for baseline fat%. e Basic model with additional adjustment for change in energy intake. f Non-linear association, p = 0.02. Beta in categories: increased energy intake, 0.34 p = 0.5; 0-500 kcal/day decrease, 0 (reference); 500-1 000 kcal/day decrease, -0.97, p = 0.003; >1,000 kcal/day decrease, -0.90, p = 0.5. g Non-linear association, p = 0.02. Beta in categories: increased energy intake, 0.40 p = 0.2; 0-500 kcal/day decrease, 0 (reference); 500-1,000 kcal/day decrease, -0.12, p = 0.6; >1,000 kcal/day decrease, -0.28, p = 0.2. h Non-linear association, p = 0.02. Beta in categories: >5 prot% decrease, 190, p = 0.01 (EI adj, 158, p = 0.03); 0-5 prot% decrease, 44.3 p = 0.2 (EI adj, 24.9, p = 0.5); 0-5 prot% increase, 0 (reference); >5 prot% increase, 61.4, p = 0.2 (EI adj, 83.0, p = 0.08). Additive effect between randomised fat group and three groups of change in energy% intake from protein (prot%), in relation to change in weight and cholesterol. The y-axis displays the mean change of weight or cholesterol in each group, and confidence intervals (CI) correspond to CI:s derived from linear regression using the largest group (low-fat, >2 units increased prot% intake) as the reference. Beta is shown for the group with the largest deviance from the reference. ▦ basic model, ▨ additional adjustment for change in energy intake. 2.5-fold and 1.8-fold greater than the single effects of the low-fat diet. Moreover, the lower reduction in plasma triglycerides in the low-fat group compared with the high-fat group, as previously reported [bib_ref] Astrup A: Randomized, multi-center trial of two hypo-energetic diets in obese subjects:..., Petersen [/bib_ref] , was confined to participants in the low-fat group who had decreased their prot% intake. Adherence to dietary interventions was not always measured in randomised trials [bib_ref] Obesity research-limitations of methods, measurements, and medications, Simons-Morton [/bib_ref] , but some studies have indicated lower adherence and smaller differences between groups than was initially targeted [bib_ref] Atkins and other low-carbohydrate diets: hoax or an effective tool for weight..., Astrup [/bib_ref] [bib_ref] Diets with high or low protein content and glycemic index for weight-loss..., Larsen [/bib_ref] [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref]. Re-analysis of dietary intake can thus provide complementary answers to intention-to-treat analyses. In our study, we analysed dietary intake in detail by the use of data from weighed food recordings during multiple days before, during and at the end of intervention. Weighed food records are often used as the reference method for validation of dietary intake reports, but measurement error exists also in weighed food records [bib_ref] Using intake biomarkers to evaluate the extent of dietary misreporting in a..., Subar [/bib_ref] , which weakens the calculated association with outcome. The quite weak association between energy reduction and weight loss in our study indicates some random error in assessment of dietary intake. Even so, the association was highly significant in contrast to observational studies that have failed to show such association [bib_ref] The association between diet and physical activity and subsequent excess weight gain..., Summerbell [/bib_ref]. A satisfactory validity of reported dietary intake in our study was indicated by the very similar results for fat group and the reported change in fat% intake, in relation to outcomes. The analysis of dietary intake data in this study does not embrace the strengths of a randomised design, but it contributes with hypotheses of dietary fat-protein proportion that have not yet been thoroughly investigated. Interaction effect (p = 0.01) between randomised fat group and three groups of change in energy% intake from protein (prot%), in relation to change in triglyceride level. The y-axis displays the mean change of triglycerides in each group, and confidence intervals (CI) correspond to CI:s derived from linear regression using the largest group (low-fat, >2 units increased prot% intake) as the reference. β is shown for the group with the largest deviance from the reference. ▦ basic model, ▨ additional adjustment for change in energy intake. Intervention studies have previously shown a positive effect of high-fat [bib_ref] Systematic review of randomized controlled trials of lowcarbohydrate vs. low-fat/low-calorie diets in..., Hession [/bib_ref] [bib_ref] Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet, Shai [/bib_ref] [bib_ref] Comparison of the Atkins, Zone, Ornish, and LEARN diets for change in..., Gardner [/bib_ref] or highprotein diets [bib_ref] Effects of variation in protein and carbohydrate intake on body mass and..., Krieger [/bib_ref] [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] on weight loss, but the clustering of high protein with high fat intake in these studies have hindered the study of combined effects of dietary fat and protein. Our results showed that, under energy restriction, the most favourable effect on weight loss and reduction in plasma lipids was a combination of a low fat intake together with an increase in the proportion of energy from protein. These results were found despite the relatively small differences between the groups of prot% change due to the prescribed protein intake of 15 energy% from protein to all participants. The differences in change were thus relatively small and were mainly attributed to differences in baseline protein intake. One should also note that in our study, an increased prot% intake was not the result of an absolute increase in protein intake, which in contrast showed a greater weight loss for greater protein reduction. This is not surprising given the concurrent reduction in energy intake with reduced protein intake. Moreover, the positive effect on weight loss for increased prot% intake was partly explained by greater energetic reduction due to a greater decrease in absolute intake of fat and carbohydrates. In support of these results, dietary protein has been shown to decrease appetite and subsequent energy intake and to increase the thermogenic effect of food [bib_ref] The effects of high protein diets on thermogenesis, satiety and weight loss:..., Halton [/bib_ref] [bib_ref] High-protein weight-loss diets: are they safe and do they work? A review..., Eisenstein [/bib_ref] , which have been key explanations to the positive effect of high dietary protein on weight loss. Sacks et al. [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] randomised 811 men and women to one of four energy-restricted diets with different macronutrient proportion over 2 years. The effect on weight loss was very similar between the diets, but the differences in macronutrient content between the diets were smaller than targeted. Indications were that weight loss was greater in the low-fat (20 fat%), high-protein (25 prot%) diet, which is compatible with our results. Sacks et al. [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] also found a greater reduction of cholesterol in the low-fat/high-protein group compared to the high-fat (40 fat%)/high-protein (25 prot%) group. The greatest reduction, however, was shown in the low-fat (20 fat%)/low-protein (15 prot%) group. In further support of our results, the study by Sacks et al. [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] showed that the low-fat/low-protein group showed a poorer effect on triglycerides compared with the three other groups. These results from our study and the study by Sacks et al. [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] indicate that the greater decrease of triglycerides in high-fat rather than low-fat diets, as found in other studies [bib_ref] Systematic review of randomized controlled trials of lowcarbohydrate vs. low-fat/low-calorie diets in..., Hession [/bib_ref] [bib_ref] Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet, Shai [/bib_ref] [bib_ref] Comparison of the Atkins, Zone, Ornish, and LEARN diets for change in..., Gardner [/bib_ref] , may only relate to low-fat diets with a low or reduced protein intake. Whilst our intervention study was large, it lasted for only 10 weeks, and long-term dietary effects are important to address. Interestingly, intervention studies of various energyrestricted diets have consistently shown that an initial weight loss period of 6-12 months is followed by weight regain, which indicates an overall poor long-term adherence of energyrestricted diets [bib_ref] Weight loss with a low-carbohydrate, Mediterranean, or low-fat diet, Shai [/bib_ref] [bib_ref] Comparison of the Atkins, Zone, Ornish, and LEARN diets for change in..., Gardner [/bib_ref] [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] [bib_ref] Low-fat dietary pattern and weight change over 7 years: the Women's Health..., Howard [/bib_ref] [bib_ref] Reduction in the incidence of type 2 diabetes with lifestyle intervention or..., Knowler [/bib_ref]. It has therefore been argued that adherence to any energydeficient diet, and not specific dietary components, is most important for long-term success of weight loss [bib_ref] Atkins and other low-carbohydrate diets: hoax or an effective tool for weight..., Astrup [/bib_ref] [bib_ref] Comparison of weight-loss diets with different compositions of fat, protein, and carbohydrates, Sacks [/bib_ref] [bib_ref] Weight-loss diets for the prevention and treatment of obesity, Katan [/bib_ref]. However, macronutrient composition may play a role in long-term adherence through a direct association with food preference, or by maintaining adherence as a result of a positive effect on weight [bib_ref] Weight-loss diets for the prevention and treatment of obesity, Katan [/bib_ref]. Our results of positive effects on weight loss and plasma lipids by a reduction in fat intake and increased proportional protein intake are only indicative and warrant further investigation in long-term randomised controlled trials. Of particular interest is to further study whether the macronutrient change or the new macronutrient level, as absolute or proportional intake, is of most importance for advantageous effects. In conclusion, this study of obese men and women on an energy-restricted diet low or high in fat over 10 weeks showed that those on the low-fat diet who had increased the percentage energy intake from protein, achieved the greatest decline in weight and plasma lipids compared with other combinations of low-/high-fat diet and decreased/increased prot% intake. [fig] Table 1: Mean (SD) baseline level, and change, of body size, metabolic factors, and dietary intake in 585 a women and men who completed the dietary intervention and in whom data on dietary intake were [/fig] [fig] Figure 1: Mean (SD) energy intake at baseline and during the intervention in men ( □ ) and women ( ■ ). [/fig] [fig] 2: Mean a (SD) absolute change in macronutrients, and change in energy intake, by groups of change in prot% intake energy intake from protein; LF = low-fat; HF = high-fat. a Mean values were weighted for the proportion of 76% women and 24% men in the total study group. [/fig] [fig] Figure 3: Fig. 3. Interaction effect (p = 0.01) between randomised fat group and three groups of change in energy% intake from protein (prot%), in relation to change in triglyceride level. The y-axis displays the mean change of triglycerides in each group, and confidence intervals (CI) correspond to CI:s derived from linear regression using the largest group (low-fat, >2 units increased prot% intake) as the reference. β is shown for the group [/fig] [table] Table 3: Beta for the effect of dietary intake on change in body size or metabolic factor (n = 585) a [/table]

Three-year survival and recurrence after first-ever stroke: the Joinville stroke registry Background: Data estimating the recurrence and risk of death are lacking in low and middle income countries, where two thirds of the stroke burden occurs. Previously we had shown that the incidence and mortality have been decreasing over the last 18 years in Joinville, Southern Brazil. In this study, we aim to determine the recurrence rates, survival rates and the cause of death in 3 years after their first-ever incident in a urban population-based setting. Methods: From the Joinville Stroke Registry, we identified all the cases of first-ever stroke that occurred from October 2009 to September 2010. Multiple overlapping sources of information were used to ensure the completeness of case identification. Patients were followed up prospectively at regular intervals from 30-days to 3 years after the index event.Kaplan-Meir and Cox proportional hazards were used to assess the cumulative risk of death and recurrence. Results: We registered 407 first-ever stroke patients. After 3 years, 136 (33%) had died. In the first year of stroke the risk of death was 28% (95% CI, 25 to 32). Beyond the first year, approximately 3 to 5% of survivors died each year. The cumulative risk of death in ischemic stroke (IS) subtypes was 3.6 higher for cardioembolic (CE) IS (hazard ratio 3.6, 95% CI, 2.1 to 6.4; p = 0.001) and 3.3 times higher for undetermined IS (HR 3.3, 95% CI 1.9 to 5.8; p = 0.001) compared to small artery occlusion IS. Over 3 years, the overall stroke recurrence risk was 9% (35/407). We found no difference in stroke recurrence risk between IS subtypes. Cardiovascular disease was the main cause of death all follow up time.Conclusions: Compared to other cohort studies conducted between 10 and 20 years ago in high-income countries, our recurrence rates and 3-year risk of death were similar. Among IS subtypes, we confirmed that CE has highest risk of death. The most common cause of death after a first-ever stroke is cardiovascular disease. This has implications for the uptake of current secondary preventive strategies and the development of new strategies. # Background Stroke is the second most common cause of death and the third most common cause of disability-adjusted lifeyears (DALYs) worldwide in 2010 [bib_ref] Global and regional mortality from 235 causes of death for 20 age..., Lozano [/bib_ref] [bib_ref] Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions,..., Murray [/bib_ref]. Incidence rates, 30-day case-fatality data, stroke survival rates and recurrence risks are useful epidemiological data which help to understand how well our primary prevention, quality of hospital care and secondary prevention have been conducted [bib_ref] Worldwide stroke incidence and early case fatality reported in 56 population-based studies:..., Feigin [/bib_ref]. The overall burden of disease data from the World Health Organization shows stroke is still in the top 6 based on disability adjusted life-years. Therefore, a comprehensive secondary prevention campaign should target the prevention of stroke recurrence, vascular dementia and death [bib_ref] Prevalence, incidence, and factors associated with pre-stroke and post-stroke dementia: a systematic..., Pendlebury [/bib_ref] [bib_ref] Long-term outcome after ischaemic stroke/transient ischaemic attack, Hankey [/bib_ref]. Recurrence and survival rates are useful data to compare the natural history of a disease against the effect of therapeutic interventions. In community-based studies which included all major stroke types together, the 3year cumulative risk of recurrent stroke has been reported to be varied from 6 to 25% [bib_ref] Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community..., Burn [/bib_ref] [bib_ref] Long-term risk of first recurrent stroke in the Perth community stroke study, Hankey [/bib_ref] [bib_ref] Three-year survival and recurrence after stroke in Malmö, Sweden: an analysis of..., Elneihoum [/bib_ref] [bib_ref] Cause of stroke recurrence is multifactorial: patterns, risk factors, and outcomes of..., Hillen [/bib_ref]. The reported cumulative risk of death in population-based studies which included all stroke types ranged across the last three decades in high and middle-income countries. In the 1980s in the UK, the 1-year risk was 31% and the 5-year risk was 50% [bib_ref] Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community..., Burn [/bib_ref]. In the 1990s, in Australia, the 1-year risk was 38% and the 5-year risk was 60%; [bib_ref] Long-term risk of first recurrent stroke in the Perth community stroke study, Hankey [/bib_ref]. In the 2000s, in Belarus, the 1-year risk was 38% and the 5-year risk was 59% [bib_ref] Long-term outcome after stroke in Belarus: the Grodno stroke study, Kulesh [/bib_ref]. As far as we know, no prospective populationbased study has been conducted in a South American country, where the stroke burden is huge. As we had shown that the incidence of stroke, 30-day case-fatality and mortality had fallen by a third in Joinville, Southern Brazil in the last decade [bib_ref] Trends in stroke incidence, mortality and case fatality rates in, Cabral [/bib_ref] (1995 to 2005), we now aimed to know the stroke recurrence behavior in our population. Therefore, we conducted a prospective community-based study of a cohort of patients after their first-ever stroke to determine the 3-year survival rates, levels of recurrence and the major causes of death in Joinville, Brazil. # Methods ## Study design and population This is a population-based prospective cohort study. Using the Joinville Stroke Registry, an ongoing population-based registry was established in 2005. We ascertained all the cases of first-ever stroke occurring among patients living in Joinville city, Southern Brazil, between October 1, 2009, and September 30, 2010. This cohort was then followed up until September 30, 2013. On the basis of intercensus data, the estimated population of the study area was 509,743 inhabitants who lived in area of 1130 km [bib_ref] Disability-adjusted life years (DALYs) for 291 diseases and injuries in 21 regions,..., Murray [/bib_ref]. Joinville city has four hospitals, three intermediate care units and one public institutional care facility. All the hospitals have computed tomography (CT) services available on a 24-hour basis. Two thirds of the city population uses only the public health system, which consists of nine state-run health districts with 56 primary care facilities. ## Baseline assessment The cohort methods have been described elsewhere [bib_ref] Trends in stroke incidence, mortality and case fatality rates in, Cabral [/bib_ref]. In brief, we used multiple overlapping data sets including medical records from all city hospitals, death certificates, and outpatient monitoring in the state-run and private units (WHO STEPS) [bib_ref] Standard method for developing stroke registers in low-income and middle-income countries: experiences..., Truelsen [/bib_ref] [bib_ref] Comparing stroke incidence worldwide: what makes studies comparable?, Sudlow [/bib_ref]. Three research nurses discussed all the stroke cases occurring in all the hospitals with a neurologist on a daily basis. On a weekly basis, a neuroradiologist unaware of patients' symptoms analyzed all brain CT scans, magnetic resonance images and digital angiographies. Every month we analyzed all death certificates (DC) issued by the Municipal Department of Health. We initially selected all death certificates containing any references to the tenth revision of the International Classification of Diseases (ICD-10) codes related to stroke (I61 through I69) or any descriptions of cerebrovascular diseases, as well as those listing the death as being from an unknown cause (R99). The deaths of patients not identified on hospitals records were investigated through the evaluation of hospital medical charts and, when available, imaging examinations. Every week the state-run health units searched their electronic records for any stroke-related diagnoses listed in the ICD-10. This happened monthly at the institutional care facility. However, not all patients identified agreed to be admitted. Such patients were offered an appointment as outpatient (1 or 2 per month, usually minor strokes or a stroke mimics). These records were reviewed and when necessary each medical chart was late discussed with a neurologist (NLC). Private city physicians received a reminder sticker with each study contact. ## Stroke definition and cohort criteria Stroke was defined according to World Health Organization (WHO) definitions [bib_ref] Cerebrovascular disease in the community: results of a WHO collaborative study, Aho [/bib_ref]. Ischemic stroke subtypes were classified according TOAST classification [bib_ref] Classification of subtype of acute ischemic stroke. definitions for use in a..., Adams [/bib_ref]. The primary outcomes were death or stroke recurrence. We defined recurrent stroke as new stroke event, additionally requiring a period of neurological stability of ≥24 hours between index and recurrent stroke, and the exclusion of other potential causes of neurological deterioration [bib_ref] Comparing risks of death and recurrent vascular events between lacunar and non-lacunar..., Jackson [/bib_ref]. Individuals who had suffered the second stroke event were again interviewed in order to identify the subtype of the stroke recurrence. All surviving patients were followed-up through telephone calls by a research nurse of the Joinville Stroke Registry at 1, 6, 12, 24 and 36 months after the event. They also had face-toface meetings with their neurologist or general physician. We classified the causes of death using standardized diagnostic criteria for stroke [bib_ref] Cerebrovascular disease in the community: results of a WHO collaborative study, Aho [/bib_ref] , recurrent stroke [bib_ref] Comparing risks of death and recurrent vascular events between lacunar and non-lacunar..., Jackson [/bib_ref] , myocardial infarction and other vascular death, and nonvascular death (see definitions, below) [bib_ref] Adelaide stroke incidence study: declining stroke rates but many preventable cardioembolic strokes, Leyden [/bib_ref]. ## Cohort inclusion and exclusion criteria We included all the cases of residents of Joinville, regardless of age, diagnosed with any type of first-ever ischemic stroke (IS), primary intracerebral hemorrhage (PIH) or subarachnoid hemorrhage (SAH). We also included residents in the city with confirmed strokes that occurred outside the city limits. Patients experiencing a stroke for the first time who had experienced a previous transient ischemic attack (TIA), were also classified as a first-ever stroke case. We excluded TIA diagnoses, all recurrent events, and stroke patients who died in the first 24 hours of onset, as well as patients residing outside the Joinville city limits, or patients with subdural, epidural or intracerebral hemorrhage secondary to arteriovenous malformation or tumors. ## Stratification types of deaths We classified the causes of death into the 5 groups that were used in the Oxfordshire Community Stroke Project: [bib_ref] Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community..., Burn [/bib_ref] (1) deaths resulting from a first-ever stroke due to the direct effects of a brain lesion or complications of resulting immobility, including deaths from bronchopneumonia even if they occurred several years after the stroke if the stroke-related disability was thought to be contributory; (2) deaths resulting from recurrent stroke that were directly due to a brain lesion or complications of immobility; (3) deaths caused by cardiovascular events that were definitely or probably from myocardial infarction, ruptured aortic aneurysms, peripheral arterial disease, or sudden death when there was no alternative explanation; (4) deaths from nonvascular events that were not due to any stroke-related disability and included such illnesses as cancer, injuries, or suicide; and (5) deaths of undetermined cause where there was insufficient information to establish a cause. # Statistical analysis We performed descriptive statistical analysis to compare baseline characteristics using the qui-square test, the Student t test, or the Mann-Whitney test according to distribution. The Kaplan-Meier product-limit technique was used to generate survival probabilities and survival curves. The risk ratio was estimated by using the Cox proportional model and the Log-Rank test to compare survival curves among stroke types. We used the R Project for Statistical Computing program version 3.1.1. The study was approved by the ethics in research committees of the Hospital Municipal São José, the Hospital Regional, the Centro Hospitalar Unimed, the Hospital Dona Helena, and the University Hospital of Joinville Region-Univille, Brazil. # Results ## Study population We identified 727 patients with stroke from October 1, 2009 to September 30, 2010. Of those, 225 had prior strokes, 63 were TIA events, nine patients died in the first 24 hours and 23 patients moved to other cities. These 320 patients were excluded. Therefore, the final cohort sample was 407 patients, among whom 83% (336/407) suffered ischemic strokes, 11% (43/407) had primary intracerebral hemorrhages and 7% (28/407) experienced subarachnoid hemorrhages [fig_ref] Figure 1: Flowchart of stroke recurrence or death over 3 years after first-ever stroke... [/fig_ref]. The mean age of these patients at baseline was 63 ± 16 years, with a median age of 64 years. The ischemic stroke subtypes distribution by TOAST classification were: 27% (92/336) small artery occlusion (SAO); 27% (91/336) undetermined (UND); 25% (84/336) cardioembolic (CE); 17% (57/336) large artery atherothrombotic (LAA) and 4% (12/336) other determined strokes (OD). [fig_ref] Table 1: Premorbid cardiovascular risk factors and medications in patients with first ever ischemic... [/fig_ref] shows the baseline characteristics, premorbid medications and premorbid risk-factor prevalence among IS, PIH and SAH patients. ## Outcome at 3 years absolute risks for all patients Surviving patients were followed-up for a minimum of 3 years and up to 3 years and 11 months. Over 3 years of follow-up, 136 died and 35 had a recurrent stroke. [fig_ref] Table 2: Kaplan-Meier estimates of probability of survival within defined time intervals after the... [/fig_ref] and [fig_ref] Figure 2: Kaplan-Meier curve showing the 3-year probability of survival after a first-ever stroke... [/fig_ref] shows that the 3-year cumulative risk of death was 33.4% (95% CI, 28.9 to 38.3). The risk of death was greatest in the first year after stroke (24.1%; 95% CI, 20.0 to 29.0) and particularly in the first 30 days after stroke (21.6%; 95% CI, 17.7 to 25.9). Beyond the first year, approximately 3 to 5% of survivors continued to die each year. No patient was lost to follow-up. Absolute risks for subgroups As expected, stratification by the type of the first-ever stroke showed that subarachnoid hemorrhage was associated with a substantially greater number of case fatalities. The 30-day case-fatality rate was 54% (12/28) in SAH, 37% in HS and 12% (40/336) in IS patients. After the first month the cumulative risk of death for all stroke types decreased. Up to 3 years the cumulative risk of death was 54% (15/28) in SAH, 53% (23/43) in PIH and 27% (92/336) in IS. [fig_ref] Figure 3: Kaplan-Meier curve showing the 3-year probability of survival after a first-ever stroke... [/fig_ref] compares the survival probabilities between IS, PIH and SAH. Among IS sub-types, the 30-day case-fatality was 22% (20/91) in UND, 18% (15/85) in CE, 9% (1/11) in OD, 2% (2/92) in SAO and also 2% (1/57) in LAA. [fig_ref] Figure 4: Kaplan-Meier curve showing the 3-year probability of survival after first-ever large artery... [/fig_ref] compares the survival curve among IS over 3 years (other determined data was not included). Compared to small artery occlusion, cardioembolic stroke carried a 3.6 times greater risk of death (HR 3.6, 95% CI, 2.1 to 6.4; p = 0.001) and undetermined stroke had a 3.3 times greater risk of death (HR 3.3, 95% CI 1.9 to 5.8; p = 0.001). Data are shown in supplementary [fig_ref] Table 1: Premorbid cardiovascular risk factors and medications in patients with first ever ischemic... [/fig_ref]. Over three years we identified a cumulative risk of 9% (35/407) for stroke recurrence. Of those, 17% (6/35) died. We found no difference in stroke recurrence rates between IS subtypes [fig_ref] Figure 5: Kaplan-Meier survival curve showing the probability that, given survival, a patient with... [/fig_ref]. [fig_ref] Figure 6: Histogram showing the proportion of patients dying from different causes during different... [/fig_ref] shows the causes of death at different time intervals after a first-ever stroke onset. During the first 30 days, 90% of deaths were due to the direct neurological effects of the index stroke. Among 30-day survivors up to 3 years, 71% of subsequent deaths were due to the first-ever (64%) or a recurrent (7%) stroke, and 23% were due to other cardiovascular causes. Among 3year survivors, 43% of subsequent deaths were due to the first-ever (14%) or a recurrent (29%) stroke, and 7% were due to other cardiovascular causes. # Discussion We identified 407 first-ever stroke cases in 2009-10 in Joinville, Brazil. Over the next 3 years, the recurrence rate was 9%, with no difference in the recurrence risk among IS subtypes. As expected, the absolute risk of death was higher in the first 30 days (22%), decreasing to 3% to 5% in the following 3 years. Therefore, a sixth of patients died within 30 days and a third died within 3 years. The 30-day case-fatality rate consisted of more than a half of SAH patients, more than a third of PIH patients and 12% of IS patients. Despite the lower casefatality rate among IS patients, the risk of death was very distinct among IS subtypes, where CE and undetermined IS had a worse prognosis than the other IS subtypes. Indeed, the 30-day case-fatality rate was 22% in undetermined IS and 18% in CE, whereas we found 9% in other determined IS cases and 2% in SAO and LAA strokes. As in other community-based studies [bib_ref] Adelaide stroke incidence study: declining stroke rates but many preventable cardioembolic strokes, Leyden [/bib_ref] [bib_ref] Ischaemic stroke subtypes and associated risk factors: a French population based study, Bejot [/bib_ref] [bib_ref] Stroke associated with atrial fibrillation -Incidence and early outcomes in the North..., Hannon [/bib_ref] we also found CE and undetermined IS were the most lethal among IS subtypes. Three years after stroke onset, CE ischemic stroke patients had a 3.6 times higher risk of death, and undetermined IS patients had a 3.3 times higher risk of death than those with small artery occlusion (p < 0.001). The 136 deaths observed in the followup period included all causes of death. However, most of these were caused by cardiovascular complications in the brain or in the heart. Indeed, intracranial hypertension and immobility complications from the first-ever stroke, a recurrence, or an acute myocardial infarction were responsible for 90% of deaths within 30 days, 100% of those who died within a year and 50% of those who died within 3 years. As far as we know, there are no other stroke survival or recurrence cohort studies in a community-based setting from low or middle-income countries. Our results were similar to other cohorts, which included all major stroke types conducted between the 1980s and the 2000s in high-income countries [bib_ref] Long-term risk of first recurrent stroke in the Perth community stroke study, Hankey [/bib_ref] [bib_ref] Three-year survival and recurrence after stroke in Malmö, Sweden: an analysis of..., Elneihoum [/bib_ref] [bib_ref] Cause of stroke recurrence is multifactorial: patterns, risk factors, and outcomes of..., Hillen [/bib_ref] [bib_ref] Long-term outcome after stroke in Belarus: the Grodno stroke study, Kulesh [/bib_ref] [bib_ref] Trends in five-year survival and risk of recurrent stroke after first-ever stroke..., Hardie [/bib_ref] [bib_ref] Risk and cumulative risk of stroke recurrence: a systematic review and meta-analysis, Mohan [/bib_ref]. For example, in a meta-analysis with 9115 survivors from 13 studies, the pooled cumulative risk of stroke recurrence was 3.1% (95% CI, 1.7-4.4) at 30 days, 11.1% (95% CI, 9.0 -13.3) at 1 year and 26.4% (95% CI, 20.1-32.8) at 5 years [bib_ref] Early risk of recurrence by subtype of ischemic stroke in population-based incidence..., Lovett [/bib_ref]. At 3 years, our recurrence rate was 8.6% (95% CI, 6.1-11.7). A cohort from Oxfordshire reported that the risk of early recurrent stroke was highest in patients with LAA, a finding that supports the need for urgent carotid imaging and prompt endarterectomy [bib_ref] Early risk of recurrence by subtype of ischemic stroke in population-based incidence..., Lovett [/bib_ref]. However, as in other studies we did not find a higher pattern of stroke recurrence among major stroke types or IS subtypes [bib_ref] Three-year survival and recurrence after stroke in Malmö, Sweden: an analysis of..., Elneihoum [/bib_ref] [bib_ref] Long-term outcome after stroke in Belarus: the Grodno stroke study, Kulesh [/bib_ref] [bib_ref] Adelaide stroke incidence study: declining stroke rates but many preventable cardioembolic strokes, Leyden [/bib_ref] [bib_ref] Ischaemic stroke subtypes and associated risk factors: a French population based study, Bejot [/bib_ref] [bib_ref] Risk and cumulative risk of stroke recurrence: a systematic review and meta-analysis, Mohan [/bib_ref]. In Joinville, the one-year cumulative risk of death after a first-ever stroke was 28% (95% CI, 25% -32%) and 33% (95% CI, 29% -38%) after 3 years. Compared with other cohorts which also included SAH, PIH and IS in their samples, our rates are significantly lower than in Oxfordshire, UK (1-year: 44%, 95% CI 41-48%; 3-years: 50%, 95% CI, 46% -54%) [bib_ref] Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community..., Burn [/bib_ref] and in Perth, Australia (1-year: 43%, 95% CI, 38%-48%; 3-years:50%, 95% CI, 44% -54%) [bib_ref] Three-year survival and recurrence after stroke in Malmö, Sweden: an analysis of..., Elneihoum [/bib_ref]. several authors have studied prognostic factors for long-time survival rates after stroke [bib_ref] Cause of stroke recurrence is multifactorial: patterns, risk factors, and outcomes of..., Hillen [/bib_ref] [bib_ref] Frequency and predictors for the risk of stroke recurrence up to 10..., Mohan [/bib_ref]. It is well stablished that old age and severe impairment after stroke predict poor outcomes [bib_ref] Early risk of recurrence by subtype of ischemic stroke in population-based incidence..., Lovett [/bib_ref] [bib_ref] Trends of the incidence of ischemic stroke thrombolysis over seven years and..., Moro [/bib_ref]. We have not stratified our sample by stroke severity but one explanation for our lower risk of cumulative death might be related to lower patient age. The Joinville mean age was 63 (SD 16) years old, lower than 72 (SD not specified) years old in Oxfordshire and 73 (SD 13) years old and in Perth [bib_ref] Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community..., Burn [/bib_ref] [bib_ref] Long-term risk of first recurrent stroke in the Perth community stroke study, Hankey [/bib_ref] [bib_ref] Trends in stroke incidence, mortality and case fatality rates in, Cabral [/bib_ref] [bib_ref] Trends in five-year survival and risk of recurrent stroke after first-ever stroke..., Hardie [/bib_ref]. Therefore, the older age for first-ever onset in UK and Australia might contribute to a worse death rate in these populations, albeit the CI rates overlapped with our sample. Joinville has been using IS thrombolysis in most city hospitals since 2005 and in its stroke unit since 2000 [bib_ref] Trends of the incidence of ischemic stroke thrombolysis over seven years and..., Moro [/bib_ref] [bib_ref] Past, present, and future of stroke in middle-income countries: the Brazilian experience, Martins [/bib_ref]. Of course we cannot measure with accuracy how much these interventions, coupled with new drugs for secondary prevention which became available in the last 20 years, might be related to our better results. There are limitations to our study. First, no patient agreed to an autopsy. Despite the fact that we doublechecked each cause of death by phone and by death certificate analysis, we had no autopsy data. In contrast, the Hisayama cohort did an autopsy in 82% of their sample [bib_ref] Ten year recurrence after first ever stroke in a Japanese community: the..., Hata [/bib_ref]. Although the proportion of cases was not specified, the Oxfordshire and Perth cohorts reported they also carried out autopsies [bib_ref] Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community..., Burn [/bib_ref] [bib_ref] Long-term risk of first recurrent stroke in the Perth community stroke study, Hankey [/bib_ref]. Second, we believe that our data on the proportion of stroke recurrence might be underdetermined because it is possible that we missed some mild strokes that were not treated in hospitals. To avoid the capture of mild cases, we have been use the third step of WHO Stepwise surveillance criteria [bib_ref] Standard method for developing stroke registers in low-income and middle-income countries: experiences..., Truelsen [/bib_ref]. However, we believe that this strategy is inferior to the face-to-face follow-up meetings reported in Hisayama, South London, Oxfordshire, Perth, Erlangen and Melbourne studies [bib_ref] Long-term risk of recurrent stroke after a first-ever stroke. The Oxfordshire Community..., Burn [/bib_ref] [bib_ref] Long-term risk of first recurrent stroke in the Perth community stroke study, Hankey [/bib_ref] [bib_ref] Cause of stroke recurrence is multifactorial: patterns, risk factors, and outcomes of..., Hillen [/bib_ref] [bib_ref] Ten year recurrence after first ever stroke in a Japanese community: the..., Hata [/bib_ref] [bib_ref] Patterns of stroke recurrence according to subtype of first stroke event: the..., Azarpazhooh [/bib_ref]. Our strengths are the novelty of this data in low and middle-income countries. Our Registry is an ongoing state-run program so we have the ability to go back and measure the trends of recurrence and mortality risk in the near future in Joinville. [fig] Figure 1: Flowchart of stroke recurrence or death over 3 years after first-ever stroke in Joinville, Brazil. [/fig] [fig] Figure 3: Kaplan-Meier curve showing the 3-year probability of survival after a first-ever stroke among primary intracerebral hemorrhage (PIH), sub-arachnoid hemorrhage (SAH) and ischemic stroke (IS). [/fig] [fig] Figure 4: Kaplan-Meier curve showing the 3-year probability of survival after first-ever large artery atheroatherotsclerosis (LLA), cardioembolic (CE), undetermined (UND) and small artery occlusion (SAO) ischemic stroke. [/fig] [fig] Figure 5: Kaplan-Meier survival curve showing the probability that, given survival, a patient with a stroke will remain free of a recurrent stroke, stratified by IS subtype (other determined IS not included). [/fig] [fig] Figure 2: Kaplan-Meier curve showing the 3-year probability of survival after a first-ever stroke derived from Joinville Stroke Register, 2009-10. Dots on either side of the solid line indicate 95% CIs; n indicates number at risk at the beginning of each year. [/fig] [fig] Figure 6: Histogram showing the proportion of patients dying from different causes during different time intervals from the onset of their first-ever stroke. [/fig] [table] Table 1: Premorbid cardiovascular risk factors and medications in patients with first ever ischemic stroke, primary intracerebral hemorrhage and subarachnoid hemorrhage, Joinville 2009-2010 [/table] [table] Table 2: Kaplan-Meier estimates of probability of survival within defined time intervals after the index stroke [/table]

Kissing nevus of the penis: a case report and dermatoscopic findings* Divided nevus, also known as kissing nevus, is a rare variant of congenital melanocytic nevi in which there are two adjacent nevi in areas of the body that undergo embryonic cleavage. The original description of this type of lesion was on the eyelid. The location on the penis is even rarer, with only 17 case reports in the literature so far, and only one of them described the dermoscopic findings. We report the case of a patient with divided nevus of the penis and its clinical, dermoscopic and histopathological features. # Introduction Divided nevus, also known as kissing nevus, is a clinical variant of congenital melanocytic nevi in which there are two adjacent nevi that involve areas of the body that undergo embryonic cleavage. [bib_ref] Divided or kissing nevus of the penis, Hardin [/bib_ref] [bib_ref] Report of two cases and review of the literature, Wang [/bib_ref] [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] [bib_ref] Divided naevus of the penis: a hypothesis on the embryological mechanism of..., Kono [/bib_ref] The original description of this type of lesion was on the eyelids in 1908, and since then few cases have been reported. The same mechanism for melanocytic lesions was already described in other rarer locations, such as fingers and penis. [bib_ref] Divided or kissing nevus of the penis, Hardin [/bib_ref] [bib_ref] Report of two cases and review of the literature, Wang [/bib_ref] [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] [bib_ref] Divided naevus of the penis: a hypothesis on the embryological mechanism of..., Kono [/bib_ref] [bib_ref] Treatment of Divided Nevus of the Penis with Circumcision and Free Inner..., Li [/bib_ref] [bib_ref] A case of solitary mastocytoma suggesting a divided form of mast cell..., Niizawa [/bib_ref] So far, there have been 17 cases reporting lesions on the penis. [bib_ref] Treatment of Divided Nevus of the Penis with Circumcision and Free Inner..., Li [/bib_ref] We present a case of divided nevus on the penis and its dermoscopic and histopathological findings. ## Case report A 9-year-old boy was referred to the dermatology department with two dark, asymptomatic lesions on the penis. The macules were seen by his mother after balano-preputial exposure when he was six years old. The patient was previously healthy, non-cir- # Discussion The divided nevus was first described in 1908 by Van Michael Paul, who reported two melanocytic lesions in opposite margins of the upper and lower eyelid that, upon closing the eyes, looked like a single lesion. [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] [bib_ref] Treatment of Divided Nevus of the Penis with Circumcision and Free Inner..., Li [/bib_ref] In 1919, it was called divided nevus or kissing nevus by Fuchs. The first case on the penis was described by Desruelles in 1998, and since then few cases were reported in the world. [bib_ref] Divided or kissing nevus of the penis, Hardin [/bib_ref] [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] [bib_ref] Treatment of Divided Nevus of the Penis with Circumcision and Free Inner..., Li [/bib_ref] It is suggested that the lesion is initially single and that it divides during the development of the external genitalia. [bib_ref] Treatment of Divided Nevus of the Penis with Circumcision and Free Inner..., Li [/bib_ref] [bib_ref] Divided naevus of the penis: two paediatric cases with dermoscopic findings, Mendes [/bib_ref] Between the 11 th and 14 th week of gestation, two invaginations appear on the penis. [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] The glandular epithelium forms the glandular urethra The diagnosis of divided nevus is clinical, and the dermoscopy and histopathology are important ancillary tests for the diagnosis and patient follow-up. Dermoscopy has been very useful in the analysis of cutaneous and mucosal pigmented lesions, helps long-term follow-up, and avoids unnecessary biopsy and surgery. Only one publication has reported dermoscopic findings of two patients with divided nevus on the penis thus far. Large globules and compound patterns were demonstrated, formed by a peripheral pigment network and an area with central globules. [bib_ref] Divided naevus of the penis: two paediatric cases with dermoscopic findings, Mendes [/bib_ref] Similarly to the literature, this patient also had the compound pattern in both macules as seen on dermoscopy. On the histopathology, most lesions present as intradermal or compound melanocytic nevi. [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] Only one case of divided nevus on the penis with malignant transformation has been reported. The patient was diagnosed at the age of 30 years, had no family history of melanoma, and presented with a rapidly enlarging lesion and changes of pigmentation. However, the dermoscopy of the lesion was not described. [bib_ref] Kissing Melanoma or Kissing Nevus of the Penis?, Egberts [/bib_ref] Malignant melanoma of the penis is very rare and corresponds to less than 2% of all primary malignant lesions of the penis. In the majority of cases, it is localized on the glans (55%), followed by the prepuce (28%), penis shaft (9%) and urethral meatus (8%). The majority of melanoma cases of the penis occurs in the 6 th and 7 th decades of life and these lesions have a poor prognosis. Due to the potential of malignant transformation of divided nevus, we highlight the importance of follow-up of the patients with clinical and dermoscopic examination in order to speed treatment in case there is any change of the lesion. The differential diagnosis should be made with melanotic macule, fixed drug eruption and melanoma (specially in adults). [bib_ref] Divided naevus of the penis: two paediatric cases with dermoscopic findings, Mendes [/bib_ref] Considering the benign nature of divided nevus, treatment is optional. [bib_ref] Divided naevus of the penis: two paediatric cases with dermoscopic findings, Mendes [/bib_ref] The therapy should focus on functional and aesthetic results. 2,3,5 Some authors performed surgical treatment with satisfactory results. The nevi are exercised and the repair is done using a graft from labial mucosa or remaining foreskin after postectomy. [bib_ref] Divided or kissing nevus of the penis, Hardin [/bib_ref] [bib_ref] Report of two cases and review of the literature, Wang [/bib_ref] [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] [bib_ref] Treatment of Divided Nevus of the Penis with Circumcision and Free Inner..., Li [/bib_ref] [bib_ref] Divided naevus of the penis: two paediatric cases with dermoscopic findings, Mendes [/bib_ref] However, for large nevi, there is the concern of aesthetic or functional damage. For this reason, the use of Nd:YAG laser may a therapeutic alternative. [bib_ref] Divided or kissing nevus of the penis, Hardin [/bib_ref] [bib_ref] Report of two cases and review of the literature, Wang [/bib_ref] [bib_ref] Kissing nevus of the penis, Yun [/bib_ref] [bib_ref] Treatment of Divided Nevus of the Penis with Circumcision and Free Inner..., Li [/bib_ref] The proper knowledge about this nevus in combination with the dermoscopic features enables patient follow-up without surgical treatment, specially in case of children, to avoid aesthetic and functional impairment resulting from a scar or a surgical defect. In our case, the patient is undergoing dermatologic and urologic follow-up as an outpatient, with programmed dermoscopy and photographic documentation of the lesion every three months.q [fig] Figure 1, Figure 3: Photomicroscopy of the glans skin. A: Proliferation of densely pigmented melanocytes along the dermal-epidermal junction and papillary dermis (Hematoxylin & eosin, X100); B: Proliferation of densely pigmented melanocytes along the dermal-epidermal junction and forming nests in the papillary dermis (Hematoxylin & eosin, X400); C: Melanocytes with evident nuclei (arrows) (Hematoxylin & eosin, X400) and the preputial epithelium divides and forms the glans and the prepuce. Derruelles advocates that the nevus is caused by the migration of melanoblasts before completing the invagination of the preputial epithelium and that after completing the embryonic division each nevus continues to develop independently. Kono suggests that the migration of melanoblasts occurs soon after the embryonic separation of the glans and prepuce. 2,3,4 Clinically, they are well-defined oval-shaped pigmented macules ranging from brown to black, with a smooth surface. They present as two mirrored lesions, symmetrical in relation to the coronal sulcus. 1,3 The site of predilection is almost always the dorsal An Bras Dermatol. 2017;92(5 Suppl 1):95-7. [/fig]

Type I Choledochal Cyst Complicated With Acute Hemorrhagic Pancreatitis: A Case Report Choledochal cysts rarely present with acute pancreatitis. We report a patient with type I choledochal cyst(s) who had concomitant acute frank hemorrhagic pancreatitis.A 14-year-old male noted with a history of recurrent abdominal pain, fever and jaundice. Ultrasonography (US) of abdomen at the Emergency Department depicted distended gall bladder with wall thickening. Apparently dilated intrahepatic ducts (IHDs) and fusiform dilatation of the common bile duct (CBD), and mild dilatation of the pancreatic duct were also noted, suggesting a type I choledochal cyst( ). Computed tomography (CT) demonstrated calcifications in the uncinate process of the pancreas in addition to the similar findings on US. He subsequently underwent choledochal cyst excision with a Roux-en-Y hepaticojejunostomy. After surgical treatment, he has been doing well for 3 years. ª Choledochal cyst is one of the most common congenital anomaly of the common bile duct (CBD) [bib_ref] Biliary disease in children, Goldman [/bib_ref] [bib_ref] Choledochal cyst and anomalous pancreaticobiliary ductal union in adults: radiological spectrum and..., Cha [/bib_ref]. The clinical presentations of choledochal cysts vary from patient to patient, and may depend on the patients' age. The symptoms are more subtle and protean in adulthood than in childhood [bib_ref] Choledochal cyst and anomalous pancreaticobiliary ductal union in adults: radiological spectrum and..., Cha [/bib_ref] [bib_ref] Choledochal cysts in adults and their complications, Atkinson [/bib_ref]. In children, the major manifestations include jaundice, right upper quadrant (RUQ) mass, and intermittent colic abdominal pain [bib_ref] Choledochal cysts in adults and their complications, Atkinson [/bib_ref]. Pancreatitis, although has been described in the literature, is an uncommon complication of choledochal cyst . Children may have subtle symptoms or acute pain, the pain can be related to relatively mild pancreatitis; however, acute pain caused by frank hemorrhagic pancreatitis is much rare [bib_ref] Spontaneous rupture of the bile duct associated with pancreatitis. A rare presentation, Goenka [/bib_ref]. We herein report such a case, his disease was diagnosed by using ultrasonography (US) and confirmed by computed tomography (CT) and the subsequent surgery. Case report A 14-year-old boy was brought to the emergency department (ED) by his parents, with a chief complaint of intermittent sharp abdominal pain for 2 days. According to the statement of the boy and his parents, he had past experience of frequent abdominal pain and received medication for pain control since 6 years old. Except for his episodes of abdominal pain since childhood, his past history was unremarkable: no diabetes, no hypertension, no hepatitis, no previous history of surgery or allergy. This time, he suffered from more severe sharp abdominal pain than before for two days. Due to increasing intensity of the pain, he was brought to our ED for evaluation. Physical examination revealed tenderness on the right upper quadrant of abdomen with positive Murphy's sign. His abdomen was soft on palpation with normal active bowel sounds. He had no fever, jaundice, nausea or vomiting during the past 2 days. US of abdomen at the ED was done, which depicted distended gall bladder with wall thickening. Apparently dilated intrahepatic ducts (IHDs) and fusiform dilatation of the common bile duct (CBD), and mild dilatation of the pancreatic duct were also noted, suggesting a type I choledochal cyst [fig_ref] Figure 1: Ultrasonography of the abdomen [/fig_ref]. There were evidence of splenomegaly and intraabdominal fluid accumulation in both upper and lower abdomen. L), Alk-P: 618 IU/L (normal: 104e338 IU/L). Under the impression of choledochal cyst, complicated with acute hemorrhagic pancreatitis, he was admitted to our surgical intensive care unit (SICU). CT was then done, and the diagnosis made by using US was confirmed. The pancreatic fossa was filled with hyperdense structures which were most likely due to mixed necrotic materials and blood clots [fig_ref] Figure 2: CT scan of the upper abdomen and pelvis also shows similar findings [/fig_ref]. He underwent exploratory laparotomy with total excision of CBD and gall bladder, choledochojejunostomy, and drainage of lesser sac hematoma. The surgical diagnosis was cheledochal cyst (type I) caused by congenital anomaly with APBJ1. The abnormally long common channel was 1.7 cm. After surgery, he was sent to SICU for further intensive care. The post-operative course was smooth, he was then moved to surgical ward, stayed for another 2 weeks. After discharge, he had been followed at the out-patient department with US and CT studies for more than 2 years with stable condition, and now has been doing well for 3 years. # Discussion Choledochal cyst is generally believed to be a congenital biliary disorder. It is characterized by benign cystic dilatations of the extrahepatic and/or intrahepatic biliary tree. The etiology of choledochal cyst can be multifactorial, including congenital anomaly of the biliary system and anomalous pancreatobiliary junction (APBJ) . The so-called anomalous pancreas to biliary junction is defined as a long common channel with a length greater than 1.5 cm [bib_ref] Biliary disease in children, Goldman [/bib_ref] [bib_ref] Choledochal cyst and anomalous pancreaticobiliary ductal union in adults: radiological spectrum and..., Cha [/bib_ref] [bib_ref] Choledochal cyst, El Mouhadi [/bib_ref]. The vast majority of patients with choledochal cysts have APBJ [bib_ref] Biliary disease in children, Goldman [/bib_ref] [bib_ref] Choledochal cyst and anomalous pancreaticobiliary ductal union in adults: radiological spectrum and..., Cha [/bib_ref] [bib_ref] Choledochal cyst, El Mouhadi [/bib_ref]. When there is abnormal junction between the pancreatic and biliary ducts, the pancreatic secretions and enzymes reflux into CBD, and result in inflammation and weakening of CBD, which is subsequently dilated [bib_ref] Choledochal cyst, El Mouhadi [/bib_ref]. Some other experts proposed that choledochal cyst may be part of congenital anomaly of biliary system or multiple congenital anomaly [bib_ref] Biliary disease in children, Goldman [/bib_ref] [bib_ref] Choledochal cyst, El Mouhadi [/bib_ref] [bib_ref] Fibropolycystic liver disease: CT and MR imaging findings, Brancatelli [/bib_ref]. Choledochal cyst is more prevalent in Asian than in Western countries, and more than 33% of all reported cases are from Japan. There is a female predilection, with a female-to-male ratio of 3:1e4:1. The majority (67e80%) of patients have been diagnosed before 10 years of age [bib_ref] Biliary disease in children, Goldman [/bib_ref] [bib_ref] Choledochal cyst and anomalous pancreaticobiliary ductal union in adults: radiological spectrum and..., Cha [/bib_ref] [bib_ref] Choledochal cyst, El Mouhadi [/bib_ref]. They are classified into 5 types according to Todani [bib_ref] Choledochal cyst: a 14 year surgical experience with 36 patients, Tan [/bib_ref] , i.e., 1. fusiform dilatation of the CBD; 2. diverticulum of the CBD; 3. Choledococele; 4. multiple segmental dilations of the biliary tree including CBD; and 5. Caroli disease. The classical presentation of choledochal cysts is a triad of abdominal pain, jaundice and a palpable lump seen in 13e63% of patients [bib_ref] Management of complicated choledochal cysts, Lal [/bib_ref]. Presentations of choledochal cysts vary from patient to patient, and may depend on the patients' age. The symptoms are more subtle and protean in adulthood than in childhood. In children, the major manifestations include jaundice, right upper quadrant (RUQ) mass, and intermittent colicky abdominal pain. While in adults, the triad of jaundice, abdominal pain and RUQ mass is noted in only 10e20%. The most common symptom in adult is abdominal pain. Some patients have only subtle symptoms. Morbidities of choledochal cyst include: biliary stone (80%), cholangitis, liver abscess, liver cirrhosis, portal hypertension, spontaneous rupture of bile duct, and pancreatitis [2e5]. Rarely, patients with choledochal cyst may develop cholangiocarcinoma [bib_ref] Management of complicated choledochal cysts, Lal [/bib_ref] , however, the reported rate of this malignancy in patients with choledochal cysts has been as high as 9e28%, about 25e40 times of incidence compared to normal population [bib_ref] Management of complicated choledochal cysts, Lal [/bib_ref] [bib_ref] Choledochal cystic malignancies, Bismuth [/bib_ref]. Lal et al. reported a large series of complicated choledochal cysts in 144 patients over 15 years but with a low occurrence of pancreatitis (1.4%). In their study, one patient with a choledochocele had repeated episodes of acute pancreatitis which were managed successfully with a transduodenal sphincteroplasty, and another patient had chronic pancreatitis [bib_ref] A case of early bile duct cancer arising from villous adenoma in..., Lee [/bib_ref]. Swisher et al. reviewed 32 adult patients who were treated for choledochal cysts. Thirty documented episodes of pancreatitis in 18 patients (56.3%) were noted; this was seen in all types of choledochal cysts and was not related to the age, gender or race of the patient. All eight patients with an abnormal pancreaticobiliary junction developed pancreatitis as compared to only two of the six patients with normal pancreatic duct anatomy (P < 0.006). In their series, only one patient with a type I choledochal cyst had chronic pancreatitis [bib_ref] Pancreatitis associated with adult choledochal cysts, Swisher [/bib_ref]. An anomalous anatomical arrangement of the pancreaticobiliary ductal junction (long common channel more than 15 mm) allows reciprocal reflux of bile and pancreatic juices into the biliary tree, which can cause inflammation, ectasia and dilatation [bib_ref] Nationwide survey of choledochal cysts. Analysis of coexistent anomalies, complications and surgical..., Komi [/bib_ref] [bib_ref] Surgical aspect ot cystic dilatation of the bile duct. An anomalous junction..., Ono [/bib_ref]. Amylase can be found in the bile and may explain the increased incidence of cholangiocarcinoma and gall bladder carcinoma [bib_ref] Histopathologic studies of congenital dilatation of the bile duct as related to..., Oguchi [/bib_ref]. Similarly, the entry of bile into the pancreatic duct might lead to recurrent acute pancreatitis [bib_ref] Resection in chronic pancreatitis, Williamson [/bib_ref]. In search of the literature Gouda et al. identified only 11 published reports of chronic pancreatitis in a patient population treated for choledochal cysts of which six are confirmed cases of chronic calcific pancreatitis. Chronic pancreatitis is an uncommon association in patients with choledochal cysts [bib_ref] Choledochal cysts with chronic pancreatitis in adults: report of two cases with..., Gouda [/bib_ref]. Our young patient presenting acute pain caused by frank hemorrhagic pancreatitis is much rare. In summary, the cross-sectional imaging modalities such as US, CT, MRI in conjunction with MRCP, are good tools for diagnosing choledochal cyst. MRCP may be competitive to ERCP in the diagnosis of biliary system disease. In this case, we used US, CT to make a correct diagnosis of choledochal cyst. The interpreters of hepatobiliary imaging studies should bear in mind the following normograms of the biliary anatomy; i.e., the normal CBD diameter (<0.7 cm), the normal IHD diameter (<0.3 cm at hilar region), and normal length of common channel (<1.5 cm), and the types of choledochal cysts so that a diagnosis can be made correctly when abnormal CBD feature is encountered. Acute pancreatitis is a rare occurrence in patients with type I choledochal cyst(s) and only six cases have been reported in the literature. Our patient with choledochal cyst(s) associated with pancreatitis was treated surgically with uneventful clinical course. [fig] Figure 1: Ultrasonography of the abdomen. (A) Transverse scan of the liver shows dilated IHDs (arrows) in the hilar region (B) Oblique sagittal scan of the right upper abdomen shows mild distention of the gall bladder (small arrows) and apparently dilated CBD (large arrow) (C) Fluid collection in the lesser sac is noted (arrows), surrounding the lateral and inferior margins of the caudate lobe. (D) Similarly, echogenic fluid is evident in the left upper abdomen (large arrow), medial to the spleen (small arrows) (E) The pancreas is essentially in normal size (small arrows), showing mild heterogeneous in echopattern, and associated with mild dilated main pancreatic duct, suggestive of acute (chronic) pancreatitis. Large arrows Z lesser sac fluid collection. (F) Pelvic fluid collection (arrows) adjacent to the urinary bladder. [/fig] [fig] Figure 2: CT scan of the upper abdomen and pelvis also shows similar findings: (A) Dilatation of the IHDs (arrows) (B) Dilatation of the CBD (large short arrow) and relatively hyperdense fluid collection in the left upper abdomen (small arrows). Structures with soft tissue density in the pancreatic bed (large long arrow) are most likely due to mixed necrotic material and blood clots. (C) Mild distension of the gall bladder (arrow) (D) Pelvis fluid collection (arrows). [/fig]

Autocatalytic Activation of the Furin Zymogen Requires Removal of the Emerging Enzyme's N-Terminus from the Active Site Background: Before furin can act on protein substrates, it must go through an ordered process of activation. Similar to many other proteinases, furin is synthesized as a zymogen (profurin) which becomes active only after the autocatalytic removal of its auto-inhibitory prodomain. We hypothesized that to activate profurin its prodomain had to be removed and, in addition, the emerging enzyme's N-terminus had to be ejected from the catalytic cleft.Methodology/Principal Findings: We constructed and analyzed the profurin mutants in which the egress of the emerging enzyme's N-terminus from the catalytic cleft was restricted. Mutants were autocatalytically processed at only the primary cleavage site Arg-Thr-Lys-Arg 107 QAsp 108 , but not at both the primary and the secondary (Arg-Gly-Val-Thr-Lys-Arg 75 QSer 76 ) cleavage sites, yielding, as a result, the full-length prodomain and mature furins commencing from the N-terminal Asp108. These correctly processed furin mutants, however, remained self-inhibited by the constrained N-terminal sequence which continuously occupied the S9 sub-sites of the catalytic cleft and interfered with the functional activity. Further, using the in vitro cleavage of the purified prodomain and the analyses of colon carcinoma LoVo cells with the reconstituted expression of the wild-type and mutant furins, we demonstrated that a three-step autocatalytic processing including the cleavage of the prodomain at the previously unidentified Arg-Leu-Gln-Arg 89 QGlu 90 site, is required for the efficient activation of furin.Conclusions/Significance: Collectively, our results show the restrictive role of the enzyme's N-terminal region in the autocatalytic activation mechanisms. In a conceptual form, our data apply not only to profurin alone but also to a range of self-activated proteinases. # Introduction A variety of proteins, including serine proteinases and metalloproteinases, growth factors, and adhesion molecules as well as bacterial and viral pathogens, is initially synthesized as inactive precursors. Specific processing is required to transform these proproteins into biologically active proteins [bib_ref] The activation and physiological functions of the proprotein convertases, Seidah [/bib_ref]. Furin and related proprotein convertases (PCs) are specialized serine endoproteinases, which cleave the multibasic motifs R-X-R/K/ X-R to transform proproteins into biologically active proteins and peptides [bib_ref] Furin at the cutting edge: from protein traffic to embryogenesis and disease, Thomas [/bib_ref]. Seven furin-family proteases (furin, PACE4, PC1/3, PC2, PC4, PC5/6 and PC7) have been identified in humans [bib_ref] Cutting back on pro-protein convertases: the latest approaches to pharmacological inhibition, Fugere [/bib_ref]. Ubiquitously expressed furin is the most studied enzyme among all of the PCs [bib_ref] Proprotein convertases: ''master switches'' in the regulation of tumor growth and progression, Bassi [/bib_ref] [bib_ref] Proprotein convertases in tumor progression and malignancy: novel targets in cancer therapy, Khatib [/bib_ref] [bib_ref] Proprotein convertases: lessons from knockouts, Scamuffa [/bib_ref]. Structurally and functionally, furin resembles its evolutionary precursor, yeast kexin. Furin is synthesized as a preproprotein which contains a signal peptide, a prodomain, a subtilisin-like catalytic domain, a middle P domain, a cysteine-rich region, a transmembrane anchor and a cytoplasmic tail. The Nterminal 81-residue prodomain (residues Gln 27 -Arg 107 ) functions as a potent auto-inhibitor [bib_ref] Inhibitory potency and specificity of subtilase-like pro-protein convertase (SPC) prodomains, Fugere [/bib_ref] [bib_ref] Conformational analyses of a partially-folded bioactive prodomain of human furin, Bhattacharjya [/bib_ref]. Furin and other PCs require proteolytic removal of the inhibitory prodomain to become proteolytically potent enzymes [bib_ref] Furin at the cutting edge: from protein traffic to embryogenesis and disease, Thomas [/bib_ref] [bib_ref] The peptidase zymogen proregions: nature's way of preventing undesired activation and proteolysis, Lazure [/bib_ref]. No natural protein inhibitors of furin are known. The highly potent synthetic peptidic inhibitor decanoyl-Arg-Val-Lys-Argchloromethylketone (dec-RVKR-cmk) and a1-antitrypsin variant Portland are used to inhibit furin in cleavage reactions in vitro and in cell-based tests. The original a1-antitrypsin serpin is the natural inhibitor of neutrophil elastase [bib_ref] Human plasma proteinase inhibitors, Travis [/bib_ref]. After a natural mutation of the active site Met358 to Arg the mutant, called a1-antitrypsin variant Pittsburgh, becomes a potent inhibitor of thrombin [bib_ref] Antithrombin Pittsburgh: an alpha1-antitrypsin variant causing hemorrhagic disease, Lewis [/bib_ref]. The additional, genetically engineered, mutation at position 355 generates a mutant known as a1-antitrypsin variant Portland (a1-PDX). As a result of this additional mutation, a1-PDX exhibits a minimal furin cleavage motif (Arg 355 -Ile-Pro-Arg 358 -) in its reactive site loop and is a 0.5 nM inhibitor of furin [bib_ref] Inhibition of HIV-1 gp160-dependent membrane fusion by a furin-directed alpha 1-antitrypsin variant, Anderson [/bib_ref] [bib_ref] Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an..., Jean [/bib_ref]. a1-PDX inhibits furin by a slow, tight-binding mechanism and, similar to other serpins, functions as a suicide substrate inhibitor. Because of these parameters, a1-PDX forms a covalent, SDS-and heat-stable, complex with functionally active furin [bib_ref] Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an..., Jean [/bib_ref]. It has been suggested that the activation of the de novo synthesized furin occurs through a two-step autoproteolytic mechanism leading to the cleavage of its cognate N-terminal prodomain. Only a few landmarks of furin activation are known. The folded furin zymogen initially undergoes an autocatalytic excision at the primary Arg-Thr-Lys-Arg 107 QAsp 108 site [bib_ref] Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic..., Leduc [/bib_ref] [bib_ref] Regulation of PACE propeptideprocessing activity: requirement for a post-endoplasmic reticulum compartment and..., Rehemtulla [/bib_ref]. This cleavage alone, however, is not sufficient for enzymatic activation because furin remains inhibited by its non-covalently associated prodomain [bib_ref] Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of..., Creemers [/bib_ref] [bib_ref] Intracellular trafficking and activation of the furin proprotein convertase: localization to the..., Molloy [/bib_ref]. After the translocation of furin into the mildly acidic trans-Golgi network/endoplasmic system, the primary cleavage is followed by an autocatalytic cleavage at the secondary Arg-Gly-Val-Thr-Lys-Arg 75 QSer 76 site which releases the prodomain fragments [bib_ref] The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is..., Anderson [/bib_ref] [bib_ref] Activation of the furin endoprotease is a multiple-step process: requirements for acidification..., Anderson [/bib_ref]. As a result, the prodomain's inhibitory function is inactivated and the mature, proteolytically potent enzyme is generated. These and other studies [bib_ref] Identification of furin pro-region determinants involved in folding and activation, Bissonnette [/bib_ref] [bib_ref] Identification of a pH sensor in the furin propeptide that regulates enzyme..., Feliciangeli [/bib_ref] were focused on the inhibitory prodomain. The functional significance of the emerging N-terminal mature furin sequence, despite its obvious role in the autocatalytic activation mechanism, remains unexplored. We hypothesized that to render the emerging enzyme functionally active its N-terminal region had to egress from its original position in the preformed catalytic cleft of profurin. This event is in addition to the autocatalytic removal of the inhibitory prodomain. We also suspect that exhaustive proteolysis of the liberated prodomain is also required to prevent re-inhibition of the emerging, activated furin. To support our hypothesis, we constructed and analyzed mutant furins with amino acid substitutions and insertions into the N-terminal region of the emerging enzyme and re-examined the proteolytic processing of the furin prodomain. Collectively, our biochemical experiments and in silico modeling determined that an ejection mechanism is necessary to accomplish the exodus of the N-terminus from the active site of the emerging enzyme. If this mechanism is inactivated and the N-terminus is not released from the catalytic cleft, profurin will be processed but the resulting enzyme will remain self-inhibited. # Results ## Modeling of the activation mechanism In our modeled profurin structure, the relative position of the prodomain and the catalytic domain was similar to that reported for the subtilisin-propeptide complex (Protein Data Bank code 1SCJ). The identified helix sheet interaction of the propeptide with the catalytic domain is conserved in the subtilisin family [bib_ref] Proprotein convertase models based on the crystal structures of furin and kexin:..., Henrich [/bib_ref] [bib_ref] Subtilases: the superfamily of subtilisin-like serine proteases, Siezen [/bib_ref] and also in PCs including furin [fig_ref] Figure 1: Structural model of the WT and K117P furins [/fig_ref]. The 101-113 sequence region that spans the primary Arg-Thr-Lys-Arg 107 -QAsp 108 cleavage site was positioned in the active site of profurin in a manner similar to the dec-RVKR-cmk inhibitor in the 1P8J crystal structure of murine furin [bib_ref] The crystal structure of the proprotein processing proteinase furin explains its stringent..., Henrich [/bib_ref] [bib_ref] The kindest cuts of all: crystal structures of Kex2 and furin reveal..., Rockwell [/bib_ref]. According to the X-ray structure of furin, the position of the Nterminal Asp108 is ,40 Å away from the catalytic cleft [bib_ref] The crystal structure of the proprotein processing proteinase furin explains its stringent..., Henrich [/bib_ref]. In contrast, the primary cleavage site sequence Arg-Thr-Lys-Arg 107 QAsp 108 is located directly in the active site of the latent profurin. Through its electrostatic interactions with the calcium ion Asp115 plays a primary role in the active furin conformation by supporting the entire structural organization of the N-terminal Asp 108 -Val-Tyr-Gln-Glu-Pro-Thr-Asp-Pro-Lys 117 region. There are also multiple hydrophobic interactions of Pro113 with the neighboring Val213, Phe118 and Trp138 residues. Two additional charge contacts (Glu112-Lys135 and Lys117-Glu362) also play an important role in the stabilization of the N-terminal 108-117 region. Both the Glu112-Lys135 and Lys117-Glu362 interactions appear to stabilize the structure of the N-terminal region of the active furin molecule [fig_ref] Figure 1: Structural model of the WT and K117P furins [/fig_ref]. According to our modeling, the zymogen conformation leading to self-cleavage provides less favorable contacts for all charged residues. The electrostatic forces originating from broken ion bridges provide the energy that would be released as a result of the Arg-Thr-Lys-Arg 107 QAsp 108 site cleavage. This energy burst will stimulate repositioning of the novel N-terminus (Asp108) and will lead to the mature furin conformation where Asp108 is 40Å away from the active site. In the furin zymogen, the egress of the emerging enzyme's N-terminal region from the active site follows the autocatalytic cleavage of the primary cleavage sequence Arg-Thr-Lys-Arg 107 QAsp 108 . To accomplish this egress, the unwinding of the 108-117 region structure is necessary. This unwinding includes a change of the backbone conformation of Lys117, the abrogation of the electrostatic contact of Asp115 with the calcium ion and also with other auxiliary electrostatic contacts and the hydrophobic interactions involving Pro113. It is highly probable that the autocatalytic cleavage at the Arg-Thr-Lys-Arg 107 QAsp 108 site causes Asp115 to become available for electrostatic contact with the calcium ion and, as a result, reposition the backbone to its location observed in active furin. To accommodate the ligation of Asp115 to the calcium ion, the chain reverses direction and ejects the emerging N-terminus thus liberating the multiple S9 sites of the active site of the mature, processed, furin. A similar mechanism was earlier suggested for the maturation of subtilisin BPN' [bib_ref] The prosegment-subtilisin BPN' complex: crystal structure of a specific 'foldase, Gallagher [/bib_ref]. According to our modeling, the N-terminal insertions in the mature furin sequence should result in an extended conformation in which the primary cleavage site Arg-Thr-Lys-Arg 107 QAsp 108 will access the active site without breaking the electrostatic interactions of Asp115 with the calcium ion [fig_ref] Figure 1: Structural model of the WT and K117P furins [/fig_ref]. Following the cleavage at this primary site, the N-terminal part of the emerging active furin may become partially mobile, albeit incapable of achieving the normally occurring 40 Å repositioning, and, in contrast to wild-type (WT) furin, the N-terminal region 108-117 will continue to occupy the multiple S9 sub-sites of the active site cavity. In turn, in the K117P mutant the steric properties of proline will make any significant repositioning of the N-terminal region nearly impossible, thus making the structure of the 108-117 region in the activated mature furin similar to that of profurin. ## Recombinant constructs To support our modeling studies experimentally and to determine the requirements for the activation of furin, WT and mutant constructs were designed. To obtain the soluble constructs, which could be readily isolated from the medium by metalchelating chromatography, the human furin sequence was truncated at Ala574 and the truncation was then linked to a flexible 7-residue linker followed by a C-terminal His 6 tag. To increase the distance from the N-terminus to the critical Lys117 in the N-terminal region of furin, we constructed the insertion mutants. In the insertion mutants, a single Gly residue and the Gly 5 and Gly 6 peptide sequences were inserted between positions Pro113 and Thr114 of the furin sequence (G, G5 and G6 mutants, respectively). To confirm that the observed effects were not unique for the Gly-containing sequences, both the positively charged His 6 and the negatively charged Asp-Asp-Asp-Asp-Lys sequences were also inserted between Pro113 and Thr114 (H6 and D4K mutants, respectively). To determine the role of the hinge motion Lys117, the K117P mutant was also isolated. As a control, we constructed the catalytically inert D153N mutant with the substitution of the essential active site Asp153 [fig_ref] Figure 2: Constructs and the purification of soluble furin [/fig_ref]. The constructs were then expressed in Sf9 cells infected with the recombinant baculovirus. To determine the efficiency of the profurin synthesis and the processing and secretion of mature furins, we used Western blotting of the cell lysate and conditioned medium aliquots [fig_ref] Figure 2: Constructs and the purification of soluble furin [/fig_ref]. Two profurin forms, presumably preprofurin and profurin, were predominant in the cell lysates while the processed furin alone was detected in the medium samples. With the exception of the inert D153N construct, the levels of the mutants we observed in the cell lysate and medium samples were comparable with those of the WT construct. Because the autocatalysis is mandatory for furin processing and secretion, the inert D153N was efficiently synthesized but inefficiently processed and secreted. This inefficient processing and secretion made the purification of the D153N mutant exceedingly difficult. In addition, the molecular weight of the secreted mutants was similar to that of the WT construct suggesting that the mutations did not affect the efficiency of autocatalytic processing and that the resulting secretory mutants were each capable of prodomain processing at the primary cleavage site Arg-Thr-Lys-Arg 107 -QAsp 108 . ## Isolation and the n-terminal sequence of mutant furins We purified the mutant constructs to identify their N-terminal sequence and enzymatic features. In contrast to the inert D153N mutant, other mutants including the K117P, G, G5, G6, D4K and H6, were readily isolated from the medium using metal-chelating chromatography [fig_ref] Figure 2: Constructs and the purification of soluble furin [/fig_ref]. Because the properties of the H6and G6-furin mutants were very similar, the first will not be discussed in the text below. To confirm the conversion of profurin into a correctly processed, mature furin, the WT, K117P and G6 purified samples were each subjected to N-terminal microsequencing. Only one Nterminal peptide sequence (D 108 VYQEP) that corresponded to mature furin was identified, thus confirming that the WT and the mutants were processed through an autocatalytic activation pathway involving the cleavage of profurin at the conventional primary Arg-Thr-Lys-Arg 107 QAsp 108 site [bib_ref] Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic..., Leduc [/bib_ref] [bib_ref] Regulation of PACE propeptideprocessing activity: requirement for a post-endoplasmic reticulum compartment and..., Rehemtulla [/bib_ref] [bib_ref] The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is..., Anderson [/bib_ref] [bib_ref] Activation of the furin endoprotease is a multiple-step process: requirements for acidification..., Anderson [/bib_ref]. ## Furin activity assays The WT construct exhibited a high proteolytic activity against the Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO 2 )-Asp-OH [fig_ref] Figure 3: Characterization of mutant furins [/fig_ref] and Pyr-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide peptide substrates (data not shown). A specific inhibitor of furin (dec-RVKR-cmk) fully blocked the substrate cleavage by the WT enzyme. The correctly processed insertion mutants demonstrated low specific activity against the peptide substrates. A 1000-fold increase in the levels of the mutants in the cleavage reactions relative to that of the WT furin was required to detect their measurable activity. To corroborate our observations, we tested the ability of the mutants to specifically cleave anthrax protective antigen-83 (PA83), a target that is highly sensitive to furin proteolysis [bib_ref] Substrate cleavage analysis of furin and related proprotein convertases. A comparative study, Remacle [/bib_ref] [bib_ref] Anthrax toxin: receptor binding, internalization, pore formation, and translocation, Young [/bib_ref]. PA83 is known to be cleaved by furin at the Arg-Lys-Lys-Arg 196 site [bib_ref] Crystal structure of the anthrax toxin protective antigen, Petosa [/bib_ref] [bib_ref] Human furin is a calcium-dependent serine endoprotease that recognizes the sequence Arg-X-X-Arg..., Molloy [/bib_ref]. This cleavage converts PA83 into the PA63 functionally active species. Here, PA83 was subjected to proteolysis by increasing concentrations of the purified furins. PA83 was efficiently cleaved by the WT furin at an enzymesubstrate molar ratio as low as 1:10,000 [fig_ref] Figure 3: Characterization of mutant furins [/fig_ref]. A furin inhibitor dec-RVKR-cmk fully blocked the proteolysis of PA83 by furin. In contrast to the WT, the insertion mutants demonstrated very low activity against PA83. According to our tests, the mutants were 1,000-10,000-fold less potent against PA83 when compared to the WT enzyme. Based on these results we hypothesized that, after the prodomain excision the mutants remained self-inhibited by the N-terminal region of the mature enzyme which still occupied their active site. ## Interactions of furins with a1-pdx Normally, a1-PDX forms a covalent, SDS-and heat-stable, complex with functionally active furin [bib_ref] Antitrypsin Portland, a bioengineered serpin highly selective for furin: application as an..., Jean [/bib_ref]. To determine if the mutants were able to form a complex with a1-PDX, we coincubated furins with a1-PDX to generate a1-PDX-furin complexes. The residual furin activity was inhibited using EDTA and SDS, and then by heating the samples at 95uC. The reactions were then analyzed both by SDS-PAGE followed by Coomassie staining and Western blotting with the MON-148 antibody against the catalytic domain of furin. [fig_ref] Figure 4: Binding of mutant furins with a1-PDX [/fig_ref] shows that a1-PDX formed a stable complex with the WT furin but not with the mutants. We next employed ELISA to analyze, in greater detail, the ability of furins to form a complex with a1-PDX [fig_ref] Figure 4: Binding of mutant furins with a1-PDX [/fig_ref]. The wells of a 96-well plate were coated with the purified furins. a1-PDX tagged with a FLAG tag was then added to the wells. The levels of the bound a1-PDX were then measured using the FLAG antibody. We determined that the ability of the mutant constructs to interact with a1-PDX was comparable with that of the WT furin. These data suggested that because the multiple S sub-sites were available in the mutants they were capable of a complex formation with the a1-PDX serpin but because the abnormally positioned N-terminal sequence shielded and incapacitated the S9 sub-sites, the inactive were unable to transform the initial association with the serpin into a kinetically trapped SDS-stable complex. ## Tryptic proteolysis of the furin constructs To demonstrate that the N-terminal sequence is buried in the furin's catalytic cleft of the K117P mutant and exposed in WT furin, we used mild tryptic digestion. We suggested that the Nterminal peptides will be efficiently released by trypsin in WT furin but not in the K117P mutant. In turn, the release of the Cterminal peptides will be equally efficient in the WT and K117P constructs. The tryptic digest of the furin constructs followed by LC-MS/MS and the differential spectral count analysis of the peptides confirmed our suggestion. Thus, the release of the Nterminal D 108 VYQEPTDPKFPQQWYLSGVTQR 130 peptide electrostatic interactions of Asp115 and a calcium ion (a blue sphere), and, in addition, by two salt bridges (Glu112-Lys135 and Lys117-Glu362). (B) The 26-107 prodomain and the 108-574 catalytic domain-P domain of furin are shown in green and grey, respectively. The modeled structure of the 108-117 sequence in profurin is shown in red while the actual structure of the 108-117 sequence in the mature WT furin enzyme is in blue. Top panels -In the structure of the WT furin, the 76-107 peptide sequence, which is generated as a result of the secondary cleavage, is shown as an orange ribbon. The arrows 1 and 2 indicate the primary and the secondary cleavage sites, respectively. Left panel, profurin; middle panel, mature furin, right panel, close-up. Bottom panels -In the K117P mutant the formation of an additional helix is necessary to eject the emerging N-terminus from the active site but it is sterically forbidden because of the adjacent Pro116. As a result, the N-end region of the mature mutant enzyme continue to occupy the S9 sub-sites leading to the inactive enzyme. The arrow 1 indicates the cleavage at the primary cleavage site alone that, as a result, generates the liberated intact prodomain (green). Left panel, profurin; middle panel, mature furin, right panel, close-up. doi:10.1371/journal.pone.0005031.g001 was 10-20 times more efficient in WT furin compared to that in the K117P mutant. To the contrary, the efficiency of the release of the C-terminal peptides including G 498 DLAIHLVSPMGTR 512 , the peptide from the central part of the furin sequence (M 226 LDGEVTDAVEAR 238 ) and many addition tryptic peptides were highly similar in the WT and K117P furins . The data summarized in confirm our conclusion that the Nterminal sequence of the emerging furin enzyme continues to occupy the catalytic cleft. As a result, this N-end sequence is protected from tryptic proteolysis, especially when compared to WT furin in which this N-terminal region is exposed and, therefore, can be easily accessed by trypsin. The residual levels of the inhibitory prodomain are associated with mutant furins To exclude the possibility that the low proteolytic activity was due to the presence of the residual levels of the inhibitory prodomain in the furin samples, we evaluated the WT and K117P, G5 and G6 purified samples by Western blotting with the MON-150 antibody against the prodomain [fig_ref] Figure 6: The presence of the residual levels of the intact prodomain in the... [/fig_ref]. The WT samples did not show the presence of the prodomain. The purified mutants contained the residual levels of the 10 kDa prodomain. The molecular mass of the prodomain correlated with its full-length 27-107 sequence indicating that while the primary Arg-Thr-Lys-Arg 107 QAsp 108 cleavage site was autocatalytically processed the secondary cleavage site (Arg-Gly-Val-Thr-Lys-Arg 75 QSer 76 ) remained intact in the mutants. An additional analysis of the fractions eluted from the metalchelating column in the course of the purification of the K117P mutant demonstrated that the prodomain was co-purified with the mature mutant enzyme [fig_ref] Figure 6: The presence of the residual levels of the intact prodomain in the... [/fig_ref]. The prodomain contamination, however, was successfully removed from the samples when the metal-chelating chromatography was performed in the presence of 1 M NaCl. We also demonstrated that the prodomain contamination did not affect the cleavage activity of the K117P mutant. Indeed, the activity of the K117P mutant samples which did and did not contain the prodomain remained similarly low [fig_ref] Figure 6: The presence of the residual levels of the intact prodomain in the... [/fig_ref]. To verify that the mutant furins exhibited the full-length 27-107 prodomain, we co-incubated the G6-furin sample with the purified WT furin. The reactions were analyzed by Western blotting with the MON-150 antibody against the prodomain. As expected, the WT furin readily cleaved the secondary cleavage site (Arg-Gly-Val-Thr-Lys-Arg 75 QSer 76 ) in the prodomain of the G6 sample and inactivated its immunoreactivity [fig_ref] Figure 6: The presence of the residual levels of the intact prodomain in the... [/fig_ref]. ## Prodomain cleavage in trans To gain additional insight into the proteolytic pathway that prevents re-inhibition of furin by the residual amount of its inhibitory prodomain [fig_ref] Figure 7: Furin proteolysis in trans of its inhibitory prodomain [/fig_ref] , we re-examined proteolysis of the prodomain sequence by furin. For this purpose, we subjected the purified individual prodomain sequence to furin proteolysis in trans [fig_ref] Figure 7: Furin proteolysis in trans of its inhibitory prodomain [/fig_ref]. Because the prodomain construct was expressed in frame with a His 6 tag sequence and a short Xpress tag, the sequence of the actual prodomain commenced from Gln27 Furin constructs. The constructs were truncated at position Ala574 and C-terminally linked to a Gly 3 -Ser-Gly 3 linker followed by a His 6 tag. To generate insertion mutants, the Gly (G), Gly-Gly-Gly-Gly-Gly (G5), Gly-Gly-Gly-Gly-Gly-Gly (G6), Asp-Asp-Asp-Asp-Lys (D4K) and His-His-His-His-His-His (H6) sequences (enlarged) were inserted between positions Pro113 and Thr114 of the furin sequence. The hinge motion Lys117 and the essential Asp153 were mutated to generate the K117P and inert D153N constructs. SP, PRO, CAT, P, Cys-rich, TM and CT, signal peptide, prodomain, catalytic domain, P domain, cysteine-rich region, transmembrane domain anchor and cytoplasmic tail, respectively. The (preprofurin numbering). In contrast to the expected single cleavage of the 13 kDa tagged prodomain at the Arg-Gly-Val-Thr-Lys-Arg 75 QSer 76 that should result in two peptide products [bib_ref] The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is..., Anderson [/bib_ref] [bib_ref] Activation of the furin endoprotease is a multiple-step process: requirements for acidification..., Anderson [/bib_ref] [bib_ref] Identification of a pH sensor in the furin propeptide that regulates enzyme..., Feliciangeli [/bib_ref] , furin proteolysis of the prodomain followed by SDS-PAGE demonstrated the existence of two cleavage sites and the presence of three distinct products with apparent molecular weights of ,10 kDa, ,5 kDa and ,3 kDa in the cleavage reactions. Dec-RVKR-cmk reversed the effect of furin and rescued the prodomain from furin proteolysis. N-terminal sequencing determined that the 5 kDa fragment commenced from Ser76 and therefore represented the 76-107 region of the prodomain. The determined N-terminal sequence (EPQVQ) suggested that the 3 kDa fragment represented the 90-107 C-terminal sequence of the prodomain [fig_ref] Figure 7: Furin proteolysis in trans of its inhibitory prodomain [/fig_ref]. ## Proteolytic processing of the prodomain in lovo cells confirms a three-step mechanism To confirm the importance of the cleavage at the tertiary cleavage site (Arg-Leu-Gln-Arg 89 QGlu 90 ) in the autocatalytic processing of profurin, we prepared the full-length WT furin and also the constructs in which the primary (R107G), the secondary (K74G/R75G) and the tertiary (R89G) cleavage sites were each inactivated by mutations. We also constructed the double (R89G/ R107G, K74G/R75G/R89G and K74G/R75G/R107G) and the triple (K74G/R75G/R89G/R107G) furin mutants in which two and three cleavage sites, respectively, were inactivated by mutations. In addition, we prepared the full-length inert D153N furin. The constructs were transiently transfected in colon carcinoma LoVo cells. We specifically selected LoVo cells for these studies because of the two frameshift mutations in the furin gene of these cells do not allow them to produce furin [bib_ref] A second mutant allele of furin in the processing-incompetent cell line, LoVo...., Takahashi [/bib_ref]. Transiently transfected cells were lysed and the lysates were analyzed by Western blotting using the antibodies against the catalytic domain and the cytoplasmic tail of furin (MON-148 and MON-139, respectively). The WT furin was efficiently activated in LoVo cells and generated the intermediate and the mature enzyme [fig_ref] Figure 7: Furin proteolysis in trans of its inhibitory prodomain [/fig_ref]. The efficiency of self-activation of the R89G mutant in which the tertiary cleavage site (Arg-Leu-Gln-Arg 89 QGlu 90 ) was inactivated, was significantly reduced compared to that of WT furin. The inert mutant (D153N), the primary (R107G) and the secondary (K74G/R75G) cleavage site mutants as well as the double and triple mutants (K74G/R75G/R89G, K74G/R75G/R107G, R89G/R107G, K74G/R75G/R89G/ R107G) were incapable of self-activation and, as a result, only the profurin form was detected in the corresponding cell lysates [fig_ref] Figure 7: Furin proteolysis in trans of its inhibitory prodomain [/fig_ref]. Based on these results, we conclude that the cleavage at the tertiary cleavage site (Arg-Leu-Gln-Arg 89 QGlu 90 ) and a three-step processing is required for the efficient autocatalytic processing and inactivation of the furin inhibitory prodomain in vivo. We suggest that this extensive, three-step processing helps the emerging furin to escape re-inhibition by the residual levels of the non-covalently associated prodomain. # Discussion To prevent unwanted protein degradation and to enable spatial and temporal regulation of proteolytic activity, proteolytic enzymes are synthesized as latent precursors [bib_ref] The peptidase zymogen proregions: nature's way of preventing undesired activation and proteolysis, Lazure [/bib_ref] [bib_ref] Proteases: Multifunctional enzymes in life and disease, Lopez-Otin [/bib_ref]. Proteolytic removal of the activation segment (which is also called the inhibitory prodomain) is required to generate a mature, functionally potent, enzyme. The prodomain size ranges from short peptide sequences to independently folded domains over 100 residues. In the multiple proteinases in which the preformed active site exists in the latent precursor, the prodomain sterically blocks the active site, and thereby prevents binding of substrates. In addition to their inhibitory role, the prodomain regions are frequently important for the folding, stability, and intracellular sorting of the latent precursor. Conversion to an active enzyme is stimulated by a change in pH that results in conversion by an autocatalytic mechanism. The requirement of the autocatalytic mechanism is well established for furin, the ubiquitous proprotein convertase that plays a highly significant role in the proteolytic processing of numerous functionally important proteins and peptides. Similar mechanisms are involved in the self-activation of PCs distinct from furin. The complexity of subcellular trafficking and the role of the associated factors (eg. heparan sulfate proteoglycan) in the regulation of PC activation, however, are not completely understood [bib_ref] The regulated cell surface zymogen activation of the proprotein convertase PC5A directs..., Mayer [/bib_ref]. Generally, activation of furin requires several steps subsequent to the initial cleavage of its inhibitory N-terminal propeptide. As a result of these cleavages, the propeptide inhibitory activity is . The 500 mM imidazole elution buffer did (+NaCl) or did not (-NaCl) contain 1 M NaCl. The presence of 1 mM NaCl had no effect on the elution profile of the K117P mature furin (upper panel). (C) Specific activity of the purified WT and K117P furins against Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO 2 )-Asp-OH. The K117 mutant samples were purified either with or without 1 M NaCl and, therefore, the samples we tested either did or did not contain the residual levels of the prodomain (PRO). (D) The WT furin cleaves the prodomain observed in the G6-furin sample. The G6-furin sample (4 mg) which was purified in the absence of 1 M NaCl and therefore contained the residual amounts of the full-length prodomain, was co-incubated for 1 h at 37uC with purified furin (0.5 mg). The reactions were analyzed by Western blotting with the MON-150 antibody against the furin prodomain. doi:10.1371/journal.pone.0005031.g006 . Mass spectrometry analysis of tryptic peptides. The K117P mutant and WT furin were subjected to a mild tryptic digest (trypsin-furin ratio of 1:100 and 1:1000 w/w, 5 min). The digests were analyzed by LC-MS/MS. The relative yield of the representative Nterminal (D 108 VYQEPTDPKFPQQWYLSGVTQR 130 ) and the C-terminal (R 498 GDLAIHLVSPMGTR 512 ) peptides and the peptide from the central part of the furin sequence (M 226 LDGEVTDAVEAR 238 ) was determined using the differential peptide spectral count analysis. The yield of the WT peptides was taken as 100%. The data of the spectral count analysis of the selected peptides are summarized in . doi:10.1371/journal.pone.0005031.g005 inactivated and the propeptide cleavage fragments are released from the active site of the emerging active furin enzyme [bib_ref] Furin at the cutting edge: from protein traffic to embryogenesis and disease, Thomas [/bib_ref] [bib_ref] Activation of human furin precursor processing endoprotease occurs by an intramolecular autoproteolytic..., Leduc [/bib_ref] [bib_ref] Regulation of PACE propeptideprocessing activity: requirement for a post-endoplasmic reticulum compartment and..., Rehemtulla [/bib_ref] [bib_ref] Endoproteolytic cleavage of its propeptide is a prerequisite for efficient transport of..., Creemers [/bib_ref] [bib_ref] Intracellular trafficking and activation of the furin proprotein convertase: localization to the..., Molloy [/bib_ref] [bib_ref] The ordered and compartment-specfific autoproteolytic removal of the furin intramolecular chaperone is..., Anderson [/bib_ref] [bib_ref] Activation of the furin endoprotease is a multiple-step process: requirements for acidification..., Anderson [/bib_ref] [bib_ref] Identification of a pH sensor in the furin propeptide that regulates enzyme..., Feliciangeli [/bib_ref]. Earlier studies of the furin activation pathway have primarily been focused on processing the Nterminal prodomain thus leaving the structural re-arrangements of the N-terminal region of the emerging enzyme incompletely understood [bib_ref] Inhibitory potency and specificity of subtilase-like pro-protein convertase (SPC) prodomains, Fugere [/bib_ref] [bib_ref] Activation of the furin endoprotease is a multiple-step process: requirements for acidification..., Anderson [/bib_ref] [bib_ref] Identification of a pH sensor in the furin propeptide that regulates enzyme..., Feliciangeli [/bib_ref] [bib_ref] Bi-cycling the furin pathway: from TGN localization to pathogen activation and embryogenesis, Molloy [/bib_ref]. Because the atomic resolution structures of the catalytic domain of furin and the prodomain of furin-like PC1 are known [bib_ref] Proprotein convertase models based on the crystal structures of furin and kexin:..., Henrich [/bib_ref] [bib_ref] The crystal structure of the proprotein processing proteinase furin explains its stringent..., Henrich [/bib_ref] [bib_ref] Solution structure of the prohormone convertase 1 pro-domain from Mus musculus, Tangrea [/bib_ref] , we modeled the interactions of the prodomain and the catalytic domain which are characteristic for profurin and the transitions of the structure that follow the autocatalytic processing at the primary Arg-Thr-Lys-Arg 107 -QAsp 108 site. It appears from our in silico modeling that the relative position of the N-terminal region of the catalytic domain is distinct in profurin and in mature furin. Following the cleavage at the primary site Arg-Thr-Lys-Arg 107 QAsp 108 , the N-terminal region moves away from its original position in profurin to reach its final position in the mature furin molecule. According to our modeling, the electrostatic interactions of Asp115 with the calcium ion are especially important for the repositioning of the N-terminal 108-113 region. The electrostatic interactions involving Asp115 and the calcium ion perform like a ''loaded spring'' and this spring is released after the pro-region cleavage to facilitate re-positioning of the N-terminal region from its original position in the furin zymogen to its final position in the mature, processed, furin. Our modeling also suggested that the peptide insertions provide an opportunity for the N-terminal part of the emerging furin to remain largely immobile after the autocatalytic cleavage at Arg-Thr-Lys-Arg 107 QAsp 108 and to continue to occupy the active site cavity in the mutants. As a result, the release mechanism remained inactivated and, we suspect, the mutants remained self-inhibited by the mature furin N-end region which continued to occupy the S9 positions of the catalytic cleft . To investigate these suspicions and to elucidate the autoinhibitory function of the N-terminal region in greater detail, we studied the profurin mutants with the K117P mutation of the hinge motion Lys117 and the one, five and six residue insertions between positions Pro113 and Thr114. We determined that similar to the WT furin, these mutant furins were autocatalytically processed at the primary Arg-Thr-Lys-Arg 107 QAsp 108 site yielding the mature enzyme commencing from the N-terminal Asp108. In contrast to the WT construct the purified mutants, however, have low specific activity against the fluorescent peptide substrate and anthrax PA83, one of the most sensitive furin substrates. Our additional in-solution and ELISA binding assays confirmed that although the mutants, in contrast to normal furin, were incapable of forming an SDS-and heat stable complex with the a1-PDX serpin, they did bind the serpin non-covalently. Tryptic digest of WT and mutant furins followed by LC-MS/ MS and the differential peptide spectral count analysis of the tryptic fragments confirmed our suggestion that in the K117P mutant the N-terminal region of the emerging furin enzyme continued to be buried in the furin's catalytic cleft. As a result, this N-terminal sequence of the mutant was inaccessible to trypsin, especially when compared to WT furin. Our in vitro cleavage and cell-based studies also identified an autocatalytic cleavage of the prodomain at Arg-Leu-Gln-Arg 89 QGlu 90 that is in addition to the primary (Arg-Thr-Lys-Arg 107 QAsp 108 ) and secondary (Arg-Gly-Val-Thr-Lys-Arg 75 QSer 76 ) sites. Based on our results, we now believe that a three-step processing of the auto-inhibitory prodomain is required for its inactivation and for preventing re-inhibition of the emerging furin enzyme by the residual amounts of the non-covalently associated inhibitory prodomain. These results add another level of complexity to the molecular mechanics that are involved in the stepwise autocatalytic processing of the prodomain and imply a role for the auxiliary proteins and associated factors in these mechanisms [bib_ref] The regulated cell surface zymogen activation of the proprotein convertase PC5A directs..., Mayer [/bib_ref]. Collectively, our results describe the events leading to the activation of furin and shed additional light on the restrictive role which is played by the enzyme's N-terminal region in the autocatalytic activation mechanisms of this and potentially other self-activated proteinases. # Materials and methods # Materials Reagents were purchased from Sigma-Aldrich unless indicated otherwise. The fluorescent substrate pyroglutamic acid-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide was obtained from Peptides International. The internally quenched fluorogenic peptide substrate Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO2)-Asp-OH and the furin inhibitor dec-RVKR-cmk were from Bachem. Anthrax protective antigen-83 (PA83) was purchased from List Biological Laboratories. FLAG-tagged furin inhibitor a1-antitrypsin variant Portland (a1-PDX) was from Affinity BioReagents. Restriction enzymes were from New England Biolabs. Sf9 insect cell line (an ovarian cell line from fall armyworm Spodoptera frugiperda), Cellfectin Reagent and a pcDNA 3.1 Directional TOPO Expression Kit were obtained from Invitrogen. The baculovirus transfer vector pAcGP67-A and the BaculoGold Linearized DNA were from BD Biosciences-Pharmingen. The Fugene HD transfection reagent was from Roche Diagnostics. Sf9 insect cells were routinely grown in Sf-900 II SFM medium supplemented with penicillin-streptomycin (100 units/ml and 100 mg/ml, respectively) and Fungizone/amphotericin B (0.25 mg/ml; Omega). The murine MON-148, MON-150 and MON-139 monoclonal antibodies against the catalytic domain, the prodomain and the cytoplasmic tail of furin, respectively, were from Axxora. The rabbit a-tubulin antibody was from Cell Signaling Technology. The peroxidase-conjugated donkey anti-mouse and anti-rabbit IgG were from Jackson ImmunoResearch Laboratories. The TMB/M and TMB/E substrates were purchased from Chemicon. ## Cloning of human furin and plasmid construction for the baculovirus expression system Furin was amplified by PCR from a Marathon-Ready cDNA library (Clontech) with the 59-ATGGAGCTGAGGCCCTGGT- . Self-activation of proproteinases requires the relocation of the N-terminus. Schematic representation of the autolytic activation mechanism of profurin. The 1 and 2 arrows above the protein sequence indicate the primary and secondary cleavage sites. The Nterminal Asp108 is shown in red. The mutant positions are underlined. The mutants do not perform the secondary cleavage and as a result the full-length prodomain sequence is liberated. This is in contrast to the WT processing which involves both the primary and the secondary cleavages of the prodomain and, as a result, two short fragments are generated. The repositioning of N-terminus is inactivated in the K117P and the G6 mutants. doi:10.1371/journal.pone.0005031.g008 TGC-39 and 59-CTCATCAGAGGGCGCTCTGGTC-39 oligonucleotides as direct and reverse primers, respectively. The resulting 2.4 kb cDNA fragment was cloned into the HincII site of the pUC18 plasmid and then recloned into the pcDNA3-zeo plasmid pre-digested with HindIII and EcoRI. The furin cDNA was then used as a template for preparing the furin constructs. Soluble furin was truncated at the position Ala574 (numbering starts from the initiation Met-1 of pre-profurin) and then C-terminally tagged with a Gly 3 -Ser-Gly 3 linker followed by a His 6 tag. The 59-TGATAGCCCGGGCAGGGCCAGAAGGTCTTCACCAAC-ACGTGGGCTGTGCGCATC-39 and the 59-AGGTATACA-CGCGGCCGCTTAATGGTGATGATGATGGTGACCTCCC-CCAGAACCTCCCCCGGCGGTGCCATAGAGTACGAG-39 oligonucleotides were used as direct and reverse primers, respectively, in the PCR reactions (the His 6 tag sequence is underlined and the linker sequence is italicized). In the primers shown below the mutant positions are underlined. The K117P mutant (furin K117P) was generated by PCR mutagenesis using the 59-TACCAGGAGCCCACAGA-CCCCCCGTTTCCTCAGCAGTGGTACCTG-39 and 59-CA-GGTACCACTGCTGAGGAAACGGGGGGTCTGTGGGCT-CCTGGTA-39 forward and reverse primers, respectively. The D153N catalytically inert furin (furin D153N) with the substitution of the essential active site Asp153 was generated using the 59-GGCATTGTGGTCTCCATTCTGAACGATGGCATCGAG-AAGAACCAC-39 and 59-GTGGTTCTTCTCGATGCCATC-GTTCAGAATGGAGACCACAATGCC-39 forward and reverse primers, respectively. To construct the insertion mutants, the Gly (G), Gly-Gly-Gly-Gly-Gly (G5), Gly-Gly-Gly-Gly-Gly-Gly (G6), His-His-His-His-His-His (H6) and Asp-Asp-Asp-Asp-Lys (D4K) sequences were each inserted by PCR mutagenesis between the positions Pro113 and Thr114 of the furin sequence. The G mutant (G-furin) was constructed using the 59-GGGACGTGTACCAGGAGCCCGG-CACAGACCCCAAGTTTCCTCA-39 and 59-TGAGGAAACT-TGGGGTCTGTGCCGGGCTCCTGGTACACGTCCC-39 oligonucleotides as the forward and reverse primers, respectively. The 59-GGGACGTGTACCAGGAGCCCGGCGGTGGCGG-TGGCACAGACCCCAAGTTTCCTCA-39 (forward) and the 59-TGAGGAAACTTGGGGTCTGTGCCACCGCCACCGC-CGGGCTCCTGGTACACGTCCC-39 (reverse) primers were used to obtain the G5 mutant (G5-furin). The 59-GGGACGTG-TACCAGGAGCCCGGTGGCGGTGGCGGTGGCACAGA-CCCCAAGTTTCCTCA-39 (direct) and the 59-TGAGGAAAC-TTGGGGTCTGTGCCACCGCCACCGCCACCGGGCTCC-TGGTACACGTCCC-39 (reverse) primers were used to prepare the G6 mutant (G6-furin). The His6 mutant (H6-furin) was constructed using the 59-GGGACGTGTACCAGGAGCCCCA-TCACCATCATCATCACACAGACCCCAAGTTTCCTCA-39 and 59-TGAGGAAACTTGGGGTCTGTGTGATGATGATG-GTGATGGGGCTCCTGGTACACGTCCC-39 oligonucleotides as the forward and reverse primers, respectively. The 59-GG-GACGTGTACCAGGAGCCCGACGATGACGATAAGACAG-ACCCCAAGTTTCCTCA-39 (direct) and the 59-TGAGGA-AACTTGGGGTCTGTCTTATCGTCATCGTCGGGCTCC-TGGTACACGTCCC-39 (reverse) primers were used to obtain the D4K mutant (D4K-furin). The resulting constructs were cloned into the XmaI and NotI sites of the pAcGP67-A vector. The authenticity of the constructs was confirmed by DNA sequencing. ## The wild-type and mutant furin constructs expressed in colon carcinoma lovo cells The full-length human furin cDNA was sub-cloned into the pcDNA 3.1/V5-His-TOPO using the 59-CACCATGGAGCT-GAGGCCCTGGTTGCTATGGGTGGTAGCAGCAACAG-GAACCTT-39 and the 59-TCAGAGGGCGCTCTGGTCTTT-39 oligonucleotides as forward and reverse primers, respectively. The furin mutants with the mutations in the primary (Arg-Thr-Lys-Arg 107 QAsp 108 ), secondary (Arg-Gly-Val-Thr-Lys-Arg 75 QSer 76 ) and tertiary (Arg-Leu-Gln-Arg 89 QGlu 90 ) autocatalytic cleavage sites were generated by PCR mutagenesis using the wild-type furin template. The forward 59-GTGGCAAAGC-GACGGACTAAAGGGGACGTGTACCAGGAGCCCACA-39 and reverse 59-TGTGGGCTCCTGGTACACGTCCCCTT-TAGTCCGTCGCTTTGCCAC-39 primers were used to construct the primary cleavage site mutant (R107G). The forward 59-TGGCATCGAGGAGTGACGGGGGGGTCCCTGTCGCCT-CACCGC-39 and the reverse 59-GCGGTGAGGCGACAGG-GACCCCCCCGTCACTCCTCGATGCCA-39 primers were used to construct the secondary cleavage site mutant (K74G/ R75G). The forward 59-CGGCACAGCCGGCTGCAGGGGG-AGCCTCAAGTACAGTGG-39 and the reverse 59-CCACTGT-ACTTGAGGCTCCCCCTGCAGCCGGCTGTGCCG-39 primers were used to construct the tertiary cleavage site mutant (R89G). The double and triple mutants (K74G/R75G/R89G; K74G/R75G/R107G; R89G/R107G; K74G/R75G/R89G/ R107G) in which two and three cleavage sites were inactivated by mutations were obtained by routine genetic engineering manipulations. The full-length D153N catalytically inert furin with the substitution of the essential active site Asp153 was generated using the 59-GGCATTGTGGTCTCCATTCT-GAACGATGGCATCGAGAAGAACCAC-39 and 59-GTGGT-TCTTCTCGATGCCATCGTTCAGAATGGAGACCACAAT-GCC-39 forward and reverse primers, respectively. The furin mutant constructs were cloned into the pcDNA 3.1/V5-His-TOPO and their sequences were confirmed by DNA sequencing. Human colon carcinoma LoVo cells (ATCC) were grown in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum. Sub-confluent LoVo cells (5610 5 ) were transfected with 3 ml of Fugene HD reagent per 1 mg of DNA. Twenty four h after tranfection, cells were lysed in 50 mM Tris-HCl pH 8.0, supplemented with 1% SDS, 1 mM EDTA, 100 mM NaCl, 1 mM phenylmethyl sulfonylfluoride, and a protease inhibitor cocktail. The lysate samples (5 mg total protein each) were analyzed by Western blotting with the furin antibodies. ## Furin expression and purification Sf9 cells were co-transfected with the recombinant pAcGP67-A vectors and the BaculoGold linearized DNA using Cellfectin. The amplification of the recombinant baculovirus and the preparation of a high-titer viral stock were performed in Sf9 cells according to the manufacturer's instructions (Invitrogen). Cells (2610 6 cell/ml) infected at a multiplicity of infection 1-2 were then cultured in suspension at 27uC in Sf-900 II SFM medium (1 L). After a 36-38 h incubation, the medium was centrifuged at 2006g and then at 15,0006g (30 min each; 4uC). The supernatant was concentrated 20-fold using an Amicon Ultra 30K-cutoff membrane (Millipore). 1 M Tris-HCl, pH 8.0, was added to the samples to obtain a final concentration of 20 mM. Furin was isolated from the medium samples using Ni 2+chelating chromatography on the HiTrap Chelating HP 1.662.5 cm size column (Amersham Biosciences) equilibrated with PBS. After eluting the impurities with 60 ml of a linear gradient of 0-25 mM imidazole, furin was eluted with 500 mM imidazole. Where indicated, 1 M NaCl was included in all of the buffers used for the purification of furin. The purified fractions were pooled, dialyzed against PBS and stored at -80uC. Purified furin samples were analyzed by SDS-PAGE, Western blotting and enzyme activity assays. The typical yield of purified furin was approximately 1 mg/liter of cell culture medium. Following SDS-PAGE and the transfer of the proteins onto a membrane support, the N-terminal peptide sequence of furin was determined by N-terminal microsequencing at ProSeq (Boxford, MA). The SDS-and heat-stable complexes of furin with a1-PDX For the SDS-gel analysis followed by Coomassie staining, a1-PDX (5 mg, 10 mM) was co-incubated for 15 min at ambient temperature with furin (0.5 mg, 1 mM) in either PBS or the Sf-900 II SFM medium. For the Western blot analysis, the concentrations of a1-PDX and furin in the reactions were 10-fold less (0.5 mg and 50 ng, respectively). Furin activity was blocked by adding 5 mM EDTA and the 26 SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 0.005% Bromophenol Blue, 20 mM DTT and 20% glycerol) to the reactions. The reactions were heated for 3 min at 95uC and then analyzed by SDS-PAGE in a 4-20% acrylamide gradient gel followed by either Coomassie staining or Western blotting of the reaction aliquots with the MON-148 antibody. ## Elisa of the furin-a1-pdx complexes To measure the efficiency of the a1-PDX binding with furin, the wells of a 96-well plate (Nunc) were coated for 16 h at 4uC with purified furin constructs (1 mg/ml in 100 ml PBS). BSA (1 mg/ml 100 ml PBS) was used as a control. The wells were then blocked with 3% BSA in PBS-0.05% Tween 20 for 2 h at the ambient temperature. a1-PDX (50 mg/ml in 100 ml PBS containing 3% BSA and 0.05% Tween 20) was added to the wells and the plates were incubated for 1 h at the ambient temperature. After washing the wells five times with PBS-0.05% Tween 20, the murine FLAG M2 antibody (0.5 mg/ml in 100 ml PBS containing 3% BSA and 0.05% Tween 20) was added to the wells and the plates were incubated 1 h at the ambient temperature. The unbound antibody was removed by washings with PBS-0.05% Tween 20. The peroxidase-conjugated donkey anti-mouse IgG (1:10,000 dilution) was added to the wells for 1 h. Following extensive washing with PBS-0.05% Tween 20 and then with water, a TMB/E substrate was added to the wells. The reaction was stopped by adding 0.1 ml 1 M H 2 SO 4 . The A 450 value was measured using a plate reader. The samples were analyzed in triplicate. ## Purification of the furin prodomain The prodomain was prepared using PCR amplification of the full-length furin cDNA. The 59-CAGAAGGTCTTCACCAA-CACG-39 and 59-TCACCGTTTAGTCCGTCGCTTTGC-39 oligonucleotides were used as direct and reverse primers, respectively, in the PCR reactions. The amplified fragment contained the prodomain sequence from Gln27 to the Arg-Thr-Lys-Arg 107 Q primary cleavage site. The PCR fragment was subcloned into the pTrcHis A vector (Invitrogen). The resulting protein was expressed in frame with a His 6 tag sequence, a linker region and an additional Xpress short tag. One Shot TOP 10 E.coli cells (Invitrogen) were transformed with the recombinant prodomain construct. The expression of the recombinant protein was induced with 1 mM isopropyl-1-thio-b-D-galactopyranoside. The collected cells were lysed, and the recombinant prodomain was purified from the cell lysates using Ni 2+ -chelating chromatography and reverse-phase HPLC. The purity of the recombinant prodomain construct was confirmed by SDS-PAGE and mass spectrometry. ## Cleavage of the furin prodomain The furin prodomain (1.8 mg, 3.4 mM) was co-incubated with the purified furin samples (an enzyme-substrate ratio of 1:1 and 1:2) for 1 h at 37uC in 100 mM Hepes, pH 7.5, containing 1 mM CaCl 2 and 1 mM b-mercaptoethanol. Where indicated, dec-RVKR-cmk (50 mM) was added to the reactions. The molecular mass of the intact prodomain and the digest products was determined by MALDI-TOF mass spectrometry on a Brucker Daltonics Autoflex II TOF/TOF mass spectrometer. To determine the N-terminal peptide sequence of the cleavage products and, consequently, the scissile bond, the reactions were separated by Tricine/SDS-PAGE in a 10-20% polyacrylamide gradient gel followed by the transfer of the digest fragments onto a membrane support. The Coomassie stained bands were excised and their Nterminal sequence was determined by N-terminal microsequencing at ProSeq. ## Tryptic proteolysis of furin and mass spectrometry The purified WT and K117P furins (0.4 mg/ml each) were coincubated for 5 min at 37uC in with trypsin (Promega) at a trypsinfurin ratio of 1:100 and 1:1000 (w/w) in PBS. Trypsin was then inactivated by 1 mM phenylmethyl sulfonylfluoride. The digest peptides were analyzed by LC-MS/MS using an Eksigent Nano 2D-LC system coupled with LTQ mass spectrometer (Thermo Scientific). The spectra were searched using Sorcerer SEQUEST (Sage-N Research) and filtered by PeptideProphet (Institute for Systems Biology; http://peptideprophet.sourceforge.net). Only the peptides with a probability score that exceeded 0.95 and a cross-correlation (Xcorr) value that exceeded 2.0 were further considered. The yield of the tryptic peptides was measured using the differential peptide spectral count analysis [bib_ref] A model for random sampling and estimation of relative protein abundance in..., Liu [/bib_ref] [bib_ref] Correlation of relative abundance ratios derived from peptide ion chromatograms and spectrum..., Zybailov [/bib_ref]. ## Enzymatic assays Furin activity was measured in wells of a 96 well plate in 0.2 ml 100 mM Hepes, pH 7.5, containing 1 mM CaCl 2 , 1 mM bmercaptoethanol and 0.005% Brij35. Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO 2 )-Asp-OH (5 mM) was used as a substrate. The concentration of the wild-type (WT) and mutant furin in the reactions was 1 nM and 1 mM, respectively. The steady-state rate of substrate hydrolysis was monitored continuously (l ex = 320 nm and l em = 420 nm) at 37uC using a fluorescence spectrophotometer. When the Pyr-Arg-Thr-Lys-Arg-methyl-coumaryl-7-amide substrate was used, the steady-state rate was monitored at l ex = 360 nm and l em = 465 nm. The samples were measured in triplicate. ## Furin proteolysis of anthrax pa83 PA83 (1 mg, 1.2 mM) was co-incubated with furin at the indicated enzyme-substrate ratio for 1 h at 37uC in 100 mM Hepes, pH 7.5, containing 1 mM CaCl 2 and 1 mM bmercaptoethanol. The reactions were stopped by adding 26 SDS-PAGE sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 0.005% Bromophenol Blue, 20 mM DTT and 20% glycerol) to the samples. The digest reactions were analyzed in an 8-16% polyacrylamide gradient gel. ## Structure modeling The structure of the human WT and mutant profurin was modeled using the available coordinates of the highly homologous (97% identity) catalytic domain and P-domain of murine 108-574 furin (Protein Data Bank code 1P8J) and the prodomain of murine 26-107 PC1 (37% identity; Protein Data Bank code 1KN6) as templates, and the Modeler software (http://ffas.ljcrf.edu). The human furin propeptide was docked into the crystal structure of the mouse furin catalytic domain using the DOT program (www. sdcs.edu/CCMS/DOT). To avoid steric clashes the several side chain positions were corrected manually using the Xfit program (www.sdcs.edu/CCMS/Packages/XTALVIEW/xtalview.html). The structure was then energy-minimized using the CHARMm22 force field molecular dynamics software of the Discovery Studio software package (Accelrys, http://www.accelrys.com/products/ dstudio). The model was then validated using Molprobity (molprobity.biochem.duke.edu). ## Supporting information Table S1 Found at: doi:10.1371/journal.pone.0005031.s001 (0.05 MB DOC) [fig] Figure 1: Structural model of the WT and K117P furins. (A) Stereo view of the WT furin catalytic cleft before (a red ribbon) and after (a blue ribbon) the cleavage at the primary Arg-Thr-Lys-Arg 107 QAsp 108 site. The red curved arrow indicates the motion of the 108-117 sequence from its position in profurin to its final position in the mature furin enzyme. The open conformation of the mature furin enzyme is stabilized by strong [/fig] [fig] Figure 2: Constructs and the purification of soluble furin. (A) [/fig] [fig] Figure 4: Binding of mutant furins with a1-PDX. (A) Stable complex formation between a1-PDX and furin. a1-PDX (5 mg) and furins (500 ng each) were co-incubated for 15 min. The reactions were stopped by 5 mM EDTA and 2% SDS, heated for 3 min at 95uC and then a 1/10 part of each reaction was analyzed by Western blotting with the MON-148 antibody against the catalytic domain of furin (upper panel) and a 9/10 part was used in SDS-PAGE followed by Coomassie staining (lower panel). Note the presence of the kinetically trapped, SDS-resistant a1-PDX complex with the WT furin. (B) ELISA of non-covalent complexes between a1-PDX and furins. The wells of a 96-well plate were coated with purified furins. FLAG-tagged al-PDX was allowed to bind immobilized furins. The levels of the resulting complex were determined using a FLAG antibody followed by peroxidaseconjugated donkey anti-mouse IgG and a TMB/E substrate. doi:10.1371/journal.pone.0005031.g004 [/fig] [fig] Figure 3: Characterization of mutant furins. (A) Specific activity of mutant furins against Abz-Arg-Val-Lys-Arg-Gly-Leu-Ala-Tyr(NO 2 )-Asp-OH. The substrate was efficiently cleaved by the WT furin (1 nM) while the mutants (1 mM) were ,1000-less active. (B) Cleavage of anthrax PA83 by mutant furins. PA83 (1 mg) was co-incubated for 1 h at 37uC with the purified furin samples at the indicated enzyme-substrate ratio. The digest samples were analyzed by SDS-PAGE followed by Coomassie staining. Where indicated, dec-RVKR-cmk (20 mM) was added to the 1:1 molar ratio reaction to block furin activity. doi:10.1371/journal.pone.0005031.g003 [/fig] [fig] Figure 6: The presence of the residual levels of the intact prodomain in the mutant furin samples. (A) Western blotting of the purified furins. The purified furin samples (2 mg each) were analyzed by Western blotting with the MON-150 antibody against the furin prodomain. (B) The prodomain is separated from the activated K117P furin in the course of performing metal-chelating chromatography in the presence of 1 M NaCl. The K117P furin sample was purified by metal-chelating chromatography. The fractions eluted from the column using 500 mM imidazole were analyzed by Western blotting with the MON-148 antibody against the catalytic domain (upper panel) and with the MON-150 antibody against the prodomain of furin [/fig] [fig] Figure 7: Furin proteolysis in trans of its inhibitory prodomain. (A) The recombinant furin prodomain was cloned into the pTrcHis A vector and expressed in E. coli. The sequence of the furin prodomain Gln27-Arg107 is in bold. The cleavage sites are indicated by the arrows. The N-terminal peptide sequence of the prodomain starts from Gly2 because of the cleavage of the initiating Met1 in E. coli. The sequence of the cleavage products determined by N-terminal sequencing is underlined. The sequence of the Xpress tag and His 6 tag is italicized. The numbering used is for intact translated human furin. (B) The recombinant furin prodomain (1.8 mg) was incubated at 37uC for 1 h with furin at a molar ratio of 1:1 or 2:1. The digest samples were analyzed by Tricine gel electrophoresis followed by Coomassie staining. Where indicated, dec-RVKR-cmk (50 mM) was added. The sequence numbering of prodomain and cleavage products identified by MS analysis is shown on the left side. (C) The molecular mass of the intact recombinant furin prodomain and the cleavage products was determined by MALDI-TOF MS. The calculated and measured molecular mass of the peptides is shown in Da. (D) A Western blotting analysis of colon carcinoma LoVo cells transiently transfected with the WT furin (WT), the catalytically inert D153N mutant (D153N) and the furin mutants in which the primary (R107G), the secondary (K74G/R75G) and the tertiary (R89G) cleavage sites were inactivated in the prodomain sequence. The R89G/R107G; K74G/R75G/R89G and K74G/R75G/R107G, and K74G/R75G/R89G/R107G are the mutants in which two and three cleavage sites, respectively, were inactivated by mutations. Mock cells are LoVo cells transfected with the original plasmid. The antibodies to the catalytic domain and to the cytoplasmic tail of furin (MON-148 and MON-139, respectively) were used for Western blotting. To show equal loading, the samples were also analyzed by Western blotting with an a-tubulin antibody (bottom panel). doi:10.1371/journal.pone.0005031.g007 [/fig]

Associations between recent intimate partner violence and receipt and quality of perinatal health services in Uttar Pradesh ## Ueldkja es jk uke ------------------------------------------------------gs a es a la cks f/k fjlpz ,um de;q fuds 'kul dh áfrfuf/k gw ¡ a es a vkils åùkj áns 'k es a ánku dh tkus okyh loklf; ls okvks a dks le>us vks j cs grj cukus ds fy, la jfpr fd;s tk jgs ,d cms + los z {k.k es a hkkx ys us ds fy, dg jgh gw ¡ a es a 'kks /kdrkz vks a ds ,d ,s ls lew g dk áfrfuf/kro djrh gw ¡ tks åùkj çns 'k ljdkj ds lkfk feydj loklf; ls okvks a dk v/;u dj jgh gs a åùkj áns 'k es a ekr` ,oa f'k'kq loklf; dks cs grj cukus ds fy;s vxys dq n o"kks z a es a dbz loklf; dk;z øe ákjehk fd;s tk;s a xs a bl los z {k.k dk eq [; mnñs '; ekrkvks a vks j cppks a ds loklf; es a lq /kkj ykus okys bl dk;z øe ds áhkko dk vuq eku yxkuk gs a We want to know and understand the current conditions of maternal and child health and the services available to women and children in your community. We are contacting about 5,000 households in Uttar Pradesh for this survey now. There is another round of surveys planned over the next years to try understand the impact of the programs. ge leq nk;ks a es a ekrkvks a vkS j f'k'kq vks a ds LokLF; dh orZ eku fLFkfr vkS j ekr` ,oa f'k'kq LokLF; ds fy;s miyC/k LokLF; ls okvks a dks tkuuk vkS j le>uk pkgrs gS a A ge iw js ÅÙkj çns 'k ls djhc 5]000 ifjokjks a es a los Z ## {k.k djs a xs a bl los z {k.k ds ckn dk;z øe ls gks us okys áhkko dks ns [kus ds fy, vxys o"kz los z {k.k dk 1 vks j nks j gks xka We will ask questions to women who had pregnancy termination in the last two years. We would request them to answer a few questions about general household information, other family members in household, their experiences and level of satisfaction with health care related to pregnancy, delivery and newborn health, and some basic information about your household assets. The households are selected randomly to participate in the survey. The information collected from about 5,000 households from across the state will be combined in a report and research papers to discuss with the government of Uttar Pradesh and other health professionals. ge mu efgykvks a ls Á'u iw Ns a xs ftuds fiNys nks o"kZ es a xHkZ lekifr gq bZ gks A ge mu efgykvks a ls muds ifjokj dh lkekU; tkudkjh] ?kj ds vU; lnL;] xHkkZ oLFkk] Álo vkS j uotkr f'k'kq vks a ds LokLF; la ca f/kr miyC/k ls okvks a ds vuq Hko vkS j lUrq "Vh ds Lrj vkS j ikfjokfjd lEifr ds ckjs es a tkudkjh ns us dk vuq jks /k djs a xs A ifjokjks a dk p;u jS uMe fof/k ls fd;k x;k gS A iw js jkT; ls djhc 5]000 ifjokjks a ls ,d= dh xbZ lw puk dks ,df=r djds ,d fjiks VZ vkS j dq N fjlpZ is ij cuk, tk,a xs ftudh ÅÙkj çns 'k ljdkj vkS j vU; LokLF; ls ok es a dke djus okys O;fDr;ks a ds lkFk feydj ppkZ dh tk;s xhA All the answers that you/others provide will be kept privateonly survey researchers will have access to this information. You may choose not to participate in the interview. You can stop the interview at any time or ask me to clarify any question. You may also choose to withdraw from the survey at any time. ## Vkids ;k vu; mùkjnkrkvks a ds }kjk nh xbz tkudkjh xks iuh; j[kh tk;s xh & ;g tkudkjh ds oy 'kks /kdrkz vks a rd gh lhfer jgs xha vxj vki pkgs a rks vki lk{kkrdkj es a hkkx ys us ls euk dj ldrh gs a a vki lk{kkrdkj fdlh hkh le; jks d ldrh gs a ;k fdlh á'u dks le>kus ds fy;s eq >s dg ldrh gs a a vki fdlh hkh le; bl lk{kkrdkj es a hkkx ys us ls euk dj ldrh gs a a Do you have any questions for me now? D;k vki eq >s dks bZ Á'u iw Nuk pkgrh gS a \ ## Answer questions as completely as possible and proceed. tgk¡ rd lehko ç'uks a dk mùkj ns a a I have discussed with __________________ the above procedures, explicitly pointing out potential risks or discomforts. I have asked whether any questions remain and have answered these questions to the best of my ability. The subject verbally agreed to participate in this study. ## Es a us mijks dr fooj.k dks --------------------------------------dks iw jh rjg ls crk;k gs a es a us muls iw nk fd d;k muds vks j dks bz á'u gs a vks j viuh iw jh ;ks x;rk ds lkfk mu lokyks a dk tokc fn;k gs a mùkjnkrk us eks f[kd :ik ls bl v?;u ea s hkkx ys us ds fy, lgerh nh gs a ## Djs a fd d;k vks j le; lgh jgs xka urhts dk dks m doj 'khv ij fy[ks a a A. HOUSEHOLD ROSTER (RC1/2/3/4/5/6/7/8) A1 to A13 of this section will be asked to all the categories: mother of 0-5 month, mother of 6-11 month, mother of 12-23 month, woman who had an abortion/miscarriage, adolescent girl of 10-19 years, currently married women of 15-49 years, women who had still birth/death of a child, currently pregnant women of 15-49 years AID: A household member is "a person or group of people who usually live and eat from the same kitchen". Any visitor who has been living in that house and eating from the same kitchen since a while will also be included as a member of the household Lkgk;rk% ?kj ,d ;k ,d ls T;knk yks xks a dk ,s lk leq g gS tks vke rkS j ij ,d lkFk js grs gS a vkS j ,d pq This section will be asked to all the categories except adolescent girls of age 10-19 years. ## Read out to the respondent: now, i am going to ask you a few questions about your children. mÙkjnkrk dks i<+ dj lq uk;s a % vc eS a vkils vkids cPpks a ds ckjs es dq N ç'u iw Nw ¡ xhA . How many children have you given birth to till now? Please include all children that were born, whether still alive or not. Also include your children who do not currently live with you. vkius vHkh rd fdrus cPpks a dks tUe fn;k gS \ dÌ;k mu lHkh cPpks a dks 'kkfey djs a thfor tUes Fks & pkgs og vHkh thfor gS a ;k ugha A mu cPpks a dks Hkh 'kkfey djs a tks orZ eku es vkids lkFk ugha jgrs gS a A ## Record all mentioned lhkh mùkj fy[ks a a 1 = YOUR HOME viuk ?kj 2 = PARENT'S HOME ekrk&firk dk ?kj 3 = GOVERNMENT/MUNICIPAL HOSPITAL ljdkjh@uxj fuxe gLirky 4 = GOVERNMENT DISPENSARY ljdkjh fMLis a ljh 5 = UHC/UHP/UFWC ;w ,plh@;w ,pih@;w ,QMCY;w lh 6 = CHC lh,plh 7 = APHC/NPHC/BPHC ,ih,plh@,uih,plh@chih,plh 8 = SUB-CENTER mids Uae 9 = ANGANWADI CENTER vk¡ xuokM+ h ds Uae 10 = VHND xz ke LokLF; ,oa iks ".k fnol 11 = NGO HOSPITAL/CLINIC xS j ljdkjh la In which month of pregnancy did you receive IFA syrup bottles for the first time? ## Read each item below (a-h) and code yes or no for each. if 'no' go to next letter ¼,&,p½ rd i<s a + vks j iz r;s d ds fy, gk¡ a ;k ugha dk dks m djs a a ;fn ugha gs vxys v{kj ij tk;s a a गर्ात वस्था क े कौनसे मिीने में आपको पिली बार IFA हसरप की बोर्ले हमली थी? COMPLETED MONTHS iw .kZ eghus 99 DO NOT KNOW irk ugha C10 D. How many IFA syrup bottles did you receive in total during your whole pregnancy? tc cPps vkids xHkZ es a Fkk rc vkius vkbZ -,Q-,-की हसरप dh fdruh बोर्लें हमली Fkh\ . ## Number of bottles 99 do not remember ## C10e . During the whole pregnancy with [CHILD NAME], how many bottles did you consume? tc cPps vkids xHkZ es a Fkk rc vkius vkbZ -,Q-,-की हसरप dh fdruh बोर्लें खायी Fkh\ . . ## Number of bottles 99= do not remember ## C11. ask c11 if c8=1 or c10b=1 During the whole pregnancy, for how many days did you take the tablets or syrup? Are you aware about the different danger signs during pregnancy which require visit to a health facility or contacting a healthcare provider? D;k vki xHkkZ oLFkk ds nkS jku gks us okys [krjs ds fofHkUu y{k.kks a ds ckjs es a tkurh gS a ftuds fy, LokLF; ds a nz tkus dh ;k LokLF; iz nkrk ls la idZ djus dh t:jr gks rh gS \ 1 = YES gk¡ 0 = NO ugha → GO TO C11D C11 B. What are the different danger signs during pregnancy which require visit to a health facility or contacting a healthcare provider? xHkkZ oLFkk ds nkS jku [krjs ds og dkS u ls y{k.k gS a ftuds fy, LokLF; ds a nz tkus dh ;k LokLF; iz nkrk ls la idZ djus dh t:jr gks rh gS \ This section will be asked to category1 and category2, i.e. recently delivered mothers of 0-11 months child. Few of the questions will also be asked to category 3,mothers of 11-23 months child(indicated in the questions) This section will be asked to category1 and category2, i.e. recently delivered mothers of 0-11 months child. Few of the questions will also be asked to category 3,mothers of 11-23 months child(indicated in the questions) ## Probe : any others? çks c % dq n vks j\ ## Record all mentioned lhkh mùkj fy[ks a a ## Read out to the respondent the next questions ask about labor and delivery and your experience during the birth of [child name]. mÙkjnkrk dks i<+ dj lq uk;s a % vxys dq N ç'u vkids çlo vkS j ¼cPps dk uke½ ds tUe ds le; ds vuq Hkoks a ds ckjs es gS A E1. How much money had to be paid in order to get the payment/assistance? vkius foÙkh; lgk;rk@iS lk feyus ds fy, fdruh fj'or nh Fkh\ ## Total amount paid dq y nh xbz jkf'k 9999 = do not know irk ugha ## Read out to the respondent now, i'd like to ask you some questions about things that can happen during delivery and your experiences. mùkjnkrk dks i<+ dj lq uk;s a % vc es a vkils çlo ds nks jku ?kv ldus okyh ?kvuk;s a vks j vkids vuq hkoks a ds ckjs es a dq n ç'u iw nuk pkgw ¡ xha ## E18. Do you know of any danger signs or symptoms that a woman might have during delivery or within the first month after delivery that would require seeking medical care? ## D;k vki [krjs ds ,s ls y{k.kks a ;k la ds rks a ds ckjs es tkurh gs a ftudk vuq hko efgyk çlo ds nks jku ;k çlo ds ckn ,d eghus ds vunj dj ldrh gs vks j ftlds fy, fpfdrlk ns [kjs [k dh t:jr gks rh gs \ [formula] 1 = YES gk¡ 0 = NO ugha → GO TO E21 E19. [/formula] Could you name some of the danger signs or symptoms that a woman might have during delivery or within the first month after delivery that would require seeking medical care? ## D;k vki [krjs ds ,s ls y{k.kks a ;k la ds rks a ds ckjs es crk ldrh gs a ftudk vuq hko efgyk çlo ds nks jku ;k çlo ds ckn ,d eghus ds vunj dj ldrh gs vks j ftlds fy, fpfdrlk ns [kjs [k dh t:jr gks rh gs \ ## Probe : any others? çks c % dq n vks j\ ## Record all mentioned lhkh mùkj fy[ks a a ## Do not read list. code all mentions in first column. then read each item below (a-r) that wasn't mentioned and code response in second column. if not mentioned, skip to next letter. lw ph dks u i<+ s a a igys dkye es a lhkh myys [k fd;s x;s mùkjks a dks dks m djs a a blds ckn mu fcunq vks a ¼,&vkj½ dks i<+ s a ftudk myys [k ugha fd;k x;k fkk vks j nw ljs dkye es a mùkj dks dks m djs a a ;fn ugha crk;k x;k vxys fca nq ij tk;s a a [formula] 1 = YES gk¡ 0= NO ugha [/formula] ## E24. e ask only if any of the responses e22(a-r) or e23a1 is coded 1 or 2 else goto e26 ;g á'u rhkh iw ns a tc e22(a-r) es a fdlh mùkj dk dks m 1 ;k 2 gs Did you seek treatment for this problem? D;k vkius bl leL;k dk bykt djk;k Fkk\ ## Read out to the respondent the next questions are about newborn care practices after the delivery of [child name]. mùkjnkrk dks i<+ dj lq uk;s a % vxys dq n ç'u ¼cpps dk uke½ ds tue ckn uotkr f'k'kq dh ns [khkky dh çfkkvks a ds ckjs es gs a a [formula] 1 = YES gk¡ 0 = NO ugha E45a [/formula] Did the child experience asphyxia(not breathing or Gasping) during birth, not being able to cry right after birth? ## क्या जन्म क े बाद बच्चे का दम घु टा था, सास नही ीं ले पा रहा था बच्चा?(चे क कीजजये बच्चा जन्म क े तु रीं त बाद यजद रोया नही ीं था) [formula] 1 = YES gk¡ 0 = NO ugha E45b SKIP IF NO FOR E45a [/formula] Was the child resuscitated with bag and mask and established spontaneous breathing? ## क्या बच्चे को बै ग एीं ड मास्क क े मदत से साीं स वाजपस लायी गयी थी? ## E87c During the first month after birth, did the child have sepsis/pneumonia? Plain water/Boiled water lknk@mcyk ikuh c. ## जन्म क े पहले महीने में क्या बच्चे को से प्सिस/जनमोजनया हुआ था? Honey 'kgn d. Sugar/Glucose water phuh@Xyw dks t dk ikuh e. Ghutti ?kq VṼh f. Oil to clean tongue thHk lkQ djus ds fy, rs y g. Rice/Dal water Pkoy@nkyks a dk ikuh h. Commercially produced infant formula milk O;olkf;d :i ls rS ;kj f'k'kq Qkew Z yk nq /k i. Powdered/Tinned milk MCcs dk nw /k j. Cow/buffalo/goat milk xk;@HkS l@cdjh dk nw /k k. Fruit juice Qyks a dk jl l. Tea or coffee pk; ;k dkQh m. Sodas like Pepsi, Coke, Orange drink lks Mk tS ls is Ilh] dks d] ukja xh is ; n. Clear broth/ soup lw i o. Roti mixed in milk jks Vh nw /k es feykdj p. Sattu mixed in water 'kÙkw ikuh es feykdj q. Think suji/halwa iryh 'kq th@gyok r. Thin ## G5. copy vaccination date for each vaccine from the card. code '77' if card is blank and then skip to g6. write '44' in 'day' column if card shows that a vaccination was given, but no date is recorded. code 0 if date is not mentioned & the card does not show that a vaccine was given or not. if only part of date is shown on card, record '99' or '9999' for 'do not know' in the column for which information is not given. ## Vhdkdj.k dh rkjh[k d® dkmz ls fy[ks a a ;fn dkmz [kkyh gs a rks ## Skip if g2=0 Apart from these vaccines, has (CHILD NAME) ever received any other vaccine? इन टीकों क े अलावा , कर्ी बच्चे को कोई और टीका लगा ? [formula] 1 = YES gk¡ 0 = NO ugha → GO TO G12 G11 B. [/formula] Which other vaccines has (CHILD NAME) received? बच्चे को कौन से टीक े लगे थे ? (RC 1/4/6/7/8)What information related to pregnancy, delivery and child care was discussed at the VHNDs? ## Probe : any others? çks c % dq n vks j\ ## Record all mentioned lhkh mùkj fy[ks a a ## Oh-,p-,u-mh-¼xz ke loklf; ,oa iks ".k fnol½ es xhkz oklfkk] çlo ,oa cpps a dh ns [k js [k ds ckjs es fdu tkudkjh;ks a ij ppkz gq bz fkh\ ## Probe : any others? çks c % dq n vks j\ ## Record all mentioned lhkh mùkj fy[ks a a ## H. reproductive health (rc1/2/3/4/6/7) ## Read out to the respondent: the next questions are about family planning, and the various ways or methods that a couple can use to delay or avoid a pregnancy. mùkjnkrk dks i<+ dj lq uk;s a % vxys dq n ç'u ifjokj fu;ks tu ds ckjs es vks j mu rjhdka s ds ckjs es a gs ftlls ,d neifr xhkz /kkj.k dks jks dus ;k ns j djus ds fy;s iz ;ks x dj ldrs gs s a a h1. now i would like to talk about family planning -the various ways or methods that a couple can use to delay or avoid a pregnancy. which ways or methods have you heard about? vc es a vkils mu vyx vyx iz dkj ds ifjokj fu;ks tu ds mik;ks a vks j rjhdka s ds ckjs es a iw nw ¡ xh tks ,d neifr xhkz /kkj.k dks jks dus ;k ns j djus ds fy;s iz ;ks x dj ldrs gs s a vkius fdu mik;ks a vks j rjhdka s ds ckjs es a lq uk gs \ ## For methods not mentioned spontaneously, ask: have you ever heard of (method)? ftu rjhdks a ds ckjs es lo;a ugha crk;] iw ns a % d;k vkius dhkh ¼rjhds ½ ds ckjs es lq uk gs \ ## Code '1' for each method mentioned spontaneously. then proceed reading the name and description of each method not mentioned spontaneously. circle code '2' if method is recognized and code '0' if not recognized. ## Lo;a crk;s gj rjhds dks ß1þ dks m djs a a mlds ckn ftu rjhdks a dks ugha crk;k mlds uke vks j fooj.k dks i<s a + a ;fn rjhdk irk gks ß2þ dks m djs a vks j rjhdk ugha irk gks ß0þ dks m djs a a ## H24. ask only if coded 1 or 2 in h18 ;fn ,N18 es 1 ;k 2 dks M fd;k gS rc iw Ns a Before your sterilization operation, were you or your husband told that you would not be able to have any (more) children because of the operation? ## D;k ulcunh djus ls igys vki@vkids ifr dks crk;k x;k fkk fd vki vks j cpps ugha is nk dj ik;s a xh\ ## H31. ask only if the woman wants to space/limit children and is not using any method currently ds oy rhkh iw ns a ;fn efgyk cpps ugha pkgrh@cppks a es a vurj djuk pkgrh gs a vks j orz eku es ifjokj fu;ks tu dk dks bz rfjdk ugha ç;ks x dj jgh gs a You said that you do not want to have more children/want to have a space between children, why are you not using any family planning methods currently? vkius crk;k fd vki vkS j cPps ugha pkgrh@cPpks a es a vUrj djuk pkgrh gS a ] orZ eku es ifjokj fu;ks tu dk dks bZ rfjdk D;ks a ugha ç;ks x dj jgh gS a \ ## Probe : any others? çks c % dq n vks j\ ## Record all mentioned lhkh mùkj fy[ks a a ## H33. ask only if no in h32 and h32a What is the main reason that you think you will not use a contraceptive method at any time to delay or avoid in the future? vkidk Hkfo'; es cPpks a es a ns jh ;k vUrj djus ds fy, ifjokj fu;ks tu dk dks bZ rfjdk ç;ks x ugha djus dk eq [;r dkj.k D;k gS \ Which of the following things has your husband or mother-in-law ever done to oppose your using family planning? ## Code only one ## V19b How many IFA syrup bottles did you receive in total during your whole pregnancy? ## Vkbz -,q-,-की हसरप dh fdruh बोर्लें हमली fkh\ . ## Number of bottles do not remember ## V19c During the whole pregnancy, how many bottles did you consume? vkius vkbZ -,Q-,-की हसरप dh fdruh बोर्लें खायी Fkh\ . ## Number of bottles = do not remember ## V19d ask if v18>0 or v19c>0, else go to v21 Do you take the tablets and syrup daily? Were you given any anti diabetic pills for diabetes control? [formula] क्या [/formula] ## क्या आपको डायजबटीज क े इलाज़ क े जलए गोजलया दी गयी थी? SHOW ANTI DIABETIC PILLS SAMPLES 1 = YES gk¡ 0 = NO ugha ## V35p Were you given insulin injections for diabetes control? ## क्या आपको डायजबटीज क े इलाज़ क े जलए कोई सु ई लगायी गयी थी ## V37 Did you discuss plans for your delivery with [CHILD NAME's] father? 1 = YES gk¡ 0 = NO ugha D;k vkius çlo ds la ca /k es a viuh ;ks tukvks a ds ckjs es a ¼cPps dk uke½ ds firk ls fopkj&foe'kZ fd;k Fkk\ 9 = NOT APPLICABLE ykxw ugha ## V38 Did you discuss your plans for your delivery with your mother-in-law? D;k vkius çlo ds la ca /k es a viuh ;ks tukvks a ds ckjs es a viuh lkl ls fopkj&foe'kZ fd;k Fkk\ 1 = YES gk¡ 0 = NO ugha = NOT APPLICABLE ykxw ugha How to keep the baby warm cPps dks xeZ j[kuk 1 2 0 c. Skin to skin contact 'kjhj ls lVk dj j[kuk 1 2 0 d. ## Read out to the respondent: now, i would like to ask you about preparations or decisions you made for the delivery of [child name]. mùkjnkrk dks i<+ dj lq uk;s a % vc es a vkils ¼cpps dk uke½ ds tue ds ckjs es a dh xbz rs ;kfj;ks a vks j fy;s x;s qs lyks a ds ckjs es a iw nuk pkg¡ w xha ## Do not read list. code all mentions in first column. then read each item below (a-p) that wasn't mentioned and code response in second column. if not mentioned, skip to next letter. lw ph dks u i<+ s a a igys dkye es a lhkh myys [k fd;s x;s mùkjks a dks dks m djs a a blds ckn mu fcunq vks a ¼,&ih dks i<+ s a ftudk myys [k ugha fd;k x;k fkk vks j nw ljs dkye es a mùkj dks dks m djs a a ;fn ugha crk;k x;k rc vxys fca nq ij tk;s a a Not bathing the baby within 3 days after birth in case of normal baby lkekU; çlo ds ckn rhu fnuks a rd cPps dks u ugykuk 1 2 0 e. Not bathing the baby within 7 days after birth in case of low birth weight baby ;fn cPpk अल्प वजन gq vk gS lkr fnuks a rd cPps dks u ugykuk ## R. verbal autopsy (for still births/death of a child) (rc7) read out to the respondent: now, i would like to ask you some questions about when you were pregnant. mùkjnkrk dks i<+ dj lq uk;s a % vc es vkils ml le; ds ckjs es dq n ç'u iw nw ¡ xh tc vki xhkz orh fkha ## R1. During your pregnancy, did you go to see anyone for antenatal checkup? When you were pregnant, how many times did you receive antenatal checkup? tc vki xHkZ orh Fkh rc vkidh fdruh ckj Álo&iw oZ tk¡ p gq bZ Fkh\ ## No. of times bruh ckj 99 = do not know irk ugha ## R5. During your pregnancy, as part of your antenatal check-ups were any of the following done at least once? During your pregnancy, were you identified as anaemic? ## Tc vki xhkz orh fkh rc d;k vkidh álo&iw oz tk¡ p ds :i es a fueufyf[kr tk¡ p de ls de ,d ckj dh x;h fkh\ ## Read each item below (a-f) and code yes or no for each. if 'no' go to next letter ¼,&,q½ rd i<s a + vks j ár;s d ds fy, gk¡ a ;k ugha dk dks m djs a a ;fn ugha gs vxys v{kj ij tk;s a a ## जब आप गर्भ वती थी तब क्या आपको बताया गया था क े आपको खू न की कमी है ? 1 = YES gk¡ 0 = NO ugha 99 = DO NOT KNOW irk ugha R6. During your pregnancy, were you given a TT injection? tc vki xHkZ orh Fkh rks D;k vkidks fVṼul dh lq bZ yxh Fkh\ [formula] 1 = YES gk¡ 0 = NO ugha → GO TO R8 99 = DO NOT KNOW irk ugha → GO TO R8 R7. [/formula] During your pregnancy, how many times did you receive a tetanus injection? tc vki xHkZ orh Fkh rc vkidks fdrus fVṼul dh lq bZ yxh Fkh\ NO. OF TIMES fdruh ckj 99 = DO NOT KNOW/REMEMBER irk ugha @;kn ugha R8. During your pregnancy did you receive any Iron Folic Acid ## R10. How many tablets did you receive in total during your whole pregnancy? tc vki xHkZ orh Fkh rc vkidks vkbZ -,Q-,-¼rkdr dh yky xks yh½ dh fdruh xks fy;k¡ feyh Fkh\ ## Probe for whether tablets are adult (large) or pediatric (small) dose. iw ns a fd ;g xks fy;k¡ cm+ h fkha ;k nks vh fkha a ## Number of large tablets cm+ h xks fy;ks a dh la [;k NUMBER OF SMALL TABLETSNks Vh xks fy;ks a dh la [;k 999 = DO NOT REMEMBER ;kn ugha ## R11. During the whole pregnancy with [CHILD NAME], how many tablets did you consume? tc ¼cPps dk uke½ vkids xHkZ es a Fkk rc vkius vkbZ -,Q-,-¼rkdr dh yky xks yh½ dh fdruh xks fy;k¡ [kk;h Fkh\ ## Number of large tablets cm+ h xks fy;ks a dh la [;k NUMBER OF SMALL TABLETSNks Vh xks fy;ks a dh la [;k 999 = DO NOT REMEMBER ;kn ugha R12. During your pregnancy with [CHILD NAME], were you given or did you buy any iron folic syrup? tc ¼cPps dk uke½ vkids xHkZ es a Fkk] D;k vkidks vkbZ ju Qks fyd ,flM ¼rkdr½ dh flji feyh Fkh ;k vkius [kjhnh Fkh\ [formula] 1 = YES gk¡ = NO ugha → GO TO R13 0 [/formula] ## R12a In which month of pregnancy did you receive IFA syrup bottles for the first time? गर्ात वस्था क े कौनसे मिीने में आपको पिली बार IFA हसरप की बोर्ले हमली थी? ## Completed months iw .kz eghus do not know irk ugha ## R12b How many IFA syrup bottles did you receive in total during your whole pregnancy? ## Tc cpps vkids xhkz es a fkk rc vkius vkbz -,q-,-की हसरप dh fdruh बोर्लें हमली fkh\ . ## Number of bottles do not remember ## R12c During the whole pregnancy with [CHILD NAME], how many bottles did you consume? During your pregnancy, were you given or did you buy any calcium tablets? ## Tc vki xhkz orh fkh d;k vkidks ds yfl;e dh xks yh feyh fkh ;k vkius [kjhnh fkh\ [formula] 1 = YES gk¡ 0 = NO ugha → GO TO R14 [/formula] ## R13a 2 During your pregnancy, how many calcium tablets were you given or did you buy? ## Tc vki xhkz orh fkh vkidks ds yfl;e dh fdruh xks yh feyh fkh ;k vkius [kjhnh fkh\ ## Number of tablets xks fy;ks a dh la [;k 999 do not remember ;kn ugha ## R13a 3 During the whole pregnancy, how many tablets did you consume? tc vki xHkZ orh Fkh rc vkius ds yfl;e dh fdruh xks fy;k¡ [kk;h Fkh\ NUMBER OF TABLETS xks fy;ks a dh la [;k 999 = DO NOT REMEMBER ;kn ugha ## R13a 4 During the whole pregnancy, for how many days did you take the tablet? ## Tc vki xhkz orh fkh iw jh xhkkz olfk ds nks jku vkius fdrus fnuks a rd xks yh [kk;h fkh\ ## If answer is not numeric, probe for approximate number of days. ;fn mùkj la [;k es a ugha gs ] iw ns a fd fnuks a dh la [;k yxhkx d;k fkha ## No of days 999=do not remember ;kn ugha ## R13a 5 During your pregnancy with [CHILD NAME], did you take rest more frequently than when you were not pregnant? tc ¼cPps dk uke½ vkids xHkZ es a Fkk rc D;k vki xHkZ orh u gks us ds le; dh rq yuk es a vf/kd ckj vkjke djrh Fkh\ 1 = YES gk¡ 0 = NO ugha ## R13a 6 During your pregnancy with [CHILD NAME] did you consume more food as compared to when you were not pregnant? tc ¼cPps dk uke½ vkids xHkZ es a Fkk rc D;k vki xHkZ orh u gks us ds le; dh rq yuk es a vf/kd Hkks tu djrh Fkh\ [formula] 1 = YES gk¡ = NO ugha [/formula] ## R13a 7 Which of the following foods did you consume almost daily during pregnancy? कौनसे पढक े सु नाई जाने वाली चीज़ें आपने रोजाना गर्त वस्था क े दौरान खाया? ## Multiple choice 1= Dark green leafy vegetables िरर सब्ज ियां 2=Yellow or orange fruits or vegetables (e.g. Pumpkin, carrots, ripe mango, ripe papaya) पीली या नारं गी फल और सिी 3=Lentils, dal दल 4=Roti, bread or rice रोटी, चावल, ब्रे ड 5=Eggs अन्दा 6=Meat, chicken or fish मॉस 7=Milk, paneer or yogurt दू ध, पनीर, दिी 88=Othersअन्य ## R13a 8 How many kgs. did you gain during pregnancy? गर्त वस्था क े दौरान हकर्ना वजन बढ़ा? ## ___ kgs ## = do not know irk ugha ## R14. Are you aware about the different danger signs during pregnancy which require visit to a health facility or contacting a healthcare provider? D;k vki xHkkZ oLFkk ds nkS jku gks us okys [krjs ds fofHkUu y{k.kks a ds ckjs es a tkurh gS a ftuds fy, LokLF; ds a nz tkus dh ;k LokLF; Ánkrk ls la idZ djus dh t:jr gks rh gS \ 1 = YES gk¡ 0 = NO ugha → GO TO R17 ## R15. What are the different danger signs during pregnancy which require visit to a health facility or contacting a healthcare provider? xHkkZ oLFkk ds nkS jku [krjs ds og dkS u ls y{k.k gS a ftuds fy, LokLF; ds a nz tkus dh ;k LokLF; Ánkrk ls la idZ djus dh t:jr gks rh gS \ From where did you get the information about these danger signs during pregnancy? During your pregnancy with [CHILD NAME] were you told to visit a doctor/facility in case of any pregnancy complications? tc ¼cPps dk uke½ vkids xHkZ es Fkk rc D;k vkidks ;g crk;k x;k Fkk fd xHkkZ oLFkk ls la ca f/kr fdlh Hkh ijs 'kkuh ds ekeys es a MkDVj ds ikl@LokLF; ds a nz ij tk;s a \ 1 = YES gk¡ 0 = NO ugha ## Probe : any others? çks c % dq n vks j\ ## Record all mentioned ## Xhkkz olfkk ds nks jku [krjs ds y{k.kks a ds ckjs es a vkidks dgk¡ ls tkudkjh feyh fkh\ ## Probe : any others? çks c % dq n vks j\ ## Record all mentioned ## R18. During your pregnancy what kind of problems did you experience? ## Tc vki xhkz orh fkh rc vkidks fdl çdkj dh ijs 'kkuh gq bz fkh\ ## Do not read list. code all mentions in first column. then read each item below (a-m) that wasn't mentioned and code response in second column. if not mentioned, skip to next letter. lw ph dks u i<+ s a a igys dkye es a lhkh myys [k fd;s x;s mùkjks a dks dks m djs a a blds ckn mu fcunq vks a ¼,&ts ½ dks i<+ s a ftudk myys [k ugha fd;k x;k fkk vks j nw ljs dkye es a mùkj dks dks m djs a ## = yes, spontaneous gk¡ ] Lo;a crk;k 2 = YES, AFTER READING gk¡ ] i<+ us ds ckn 0 = NO ugha lw ph dks u i<+ s a A igys dkye es a lHkh mYys [k fd;s x;s mÙkjks a dks dks M djs a A blds ckn mu fcUnq vks a ¼,&ts ½ dks i<+ s a ftudk mYys [k ugha fd;k x;k Fkk vkS j nw ljs dkye es a mÙkj dks dks M djs a A ;fn ugha crk;k x;k vxys fca nq ij tk;s a A a. Vaginal bleeding ;ks uh ls jälz ko 1 2 0 b. Smelly vaginal discharge ;ks fu ls cncw nkj ikuh fudyuk 1 2 0 c. Puffy face ps gjs dk lq tu 1 2 0 d. Headache flj nnZ 1 2 0 e. Blurred vision /kq a /kyk fn[kuk 1 2 0 f. Convulsions nkS js iM+ uk 1 2 0 g. Febrile illness cq [kkj ftldk dkj.k irk ugha 1 2 0 h. Severe abdominal pain that is not labor pain is V es a rs t nnZ tks çlo ihM+ k ugha Fkh 1 2 0 i. Pallor and difficulty in breathing lk¡ l ys u es a dfBukbZ ds lkFk ps gjs dk Qhdkiu [formula] 1 2 0 j. [/formula] Any other illness dks bZ vkS j chekjh 1 2 0 R20. ## Ask only if any of the responses in r19(a-j) is coded 1 or 2, else go to r22 Did you seek treatment for this problem? D;k vkius bl leL;k dk bykt djk;k Fkk\ Did the umblical cord come out before the baby was born? 1 = YES gk¡ D;k çlo ds ckn cPPks ds ckgj fudyus ls igys uky ckgj fudyk Fkk\ 0 = NO ugha R56 Was the umbilical cord wrapped several times (more than once) around the neck of the child at birth? D;k cPPks ds tUe ds le; uky cPPks dh xnZ u ls fyiVk ¼dbZ ckj½ gq vk Fkk\ How often did this happen during the last 12 months: often, only sometimes, or not at all? fiNys ckjg eghuks a es a ,s lk dc&dc gq vk gS \ 1=OFTEN vDlj 2=SOMETIMES dHkh&dHkh 3=NEVER dHkh ugha ## Violence against women efgykvks a ds fo#) fga lk now i would like to ask you questions about violence that you might be experiencing in your household which might be perpetrated by your husband or other members of your household. vc eS a vkils dq N iz 'u iw Nw a xh ftudk la ca /k vkids ifr ;k ifjokj ds vU; lnL;ks a }kjk dh tkus okyh ml fga lk ls gS ftldk gks ldrk gS vki lkeuk dj jgh gks a A I know that some of these questions are very personal. However, your answers are crucial for helping to understand the condition of women in India. Let me assure you that your answers are completely confidential and will not be told to anyone and no one else will know that you were asked these questions. eS a tkurh gw a fd bues a ls dq N loky O;fDrxr gS a A ij Hkkjr es a efgykvks a dh fLFkfr dks le>us ds fy, vkids mÙkj egRoiw .kZ gS A eS a vkidks ;g vk'oklu ns rh gw a fd vkids mÙkj xks iuh; j[ks tk;s a xs vkS j fdlh dks Hkh ;g irk ughya pys xk fd vkils ;s iz 'u iw Ns x;s Fks A S37. First, I am going to ask you about some situations which happen to some women. Please tell me if these apply to your relationship with your (last) husband. lcls igys eS a vkils ,s lh ?kVukvks a ds ckjs es a iw Nw a xh tks fdlh Hkh efgyk ds lkFk ?kV ldrh gS a A dì;k eq >s ;g crk;s a fd D;k ;s vkids ifr ¼fiNys ifr½ ds lkFk vkids la ca /k ij ykxw gks rh gS a A P. HOUSEHOLD CHARACTERISTICS (RC1/2/3/4/5/6/7/8) This section will be asked to all categories Read out to the respondent: Now I will ask you some questions about your religion and caste. mÙkjnkrk dks i<+ dj lq uk;s a % vc eS vkils vkids /keZ vkS j tkfr ds ckjs es iw Nw ¡ xhA P1. Name of the respondent mÙkjnkrk dk uke P2. Line Number of respondent mÙkjnkrk dk ykbu ua cj P3. What is your religion? vkidk /keZ D;k gS \ TYPE ## Code one only ## P23 In the past four weeks, did your household ever run out of money to buy food? fiNys pkj lIrkg es a D;k Hkks tu [kjhnus ds fy;s vkids ikl iS lk ugha Fkk\ 1 = YES gk¡ 0 = NO ugha ## P24 In the past four weeks, did you ever limit the types of food you fed your children because you did not have enough money to buy food for a meal? fiNys pkj lIrkg es a Hkks tu [kjhnus ds fy;s i;kZ Ir iS lk u gks us ds dkj.k D;k vkidks cPps ds Hkks tu ds çdkj lhfer djuk iM+ k Fkk\

KeyPathwayMineR: De Novo Pathway Enrichment in the R Ecosystem De novo pathway enrichment is a systems biology approach in which OMICS data are projected onto a molecular interaction network to identify subnetworks representing condition-specific functional modules and molecular pathways. Compared to classical pathway enrichment analysis methods, de novo pathway enrichment is not limited to predefined lists of pathways from (curated) databases and thus particularly suited for discovering novel disease mechanisms. While several tools have been proposed for pathway enrichment, the integration of de novo pathway enrichment in end-to-end OMICS analysis workflows in the R programming language is currently limited to a single tool. To close this gap, we have implemented an R package KeyPathwayMineR (KPM-R). The package extends the features and usability of existing versions of KeyPathwayMiner by leveraging the power, flexibility and versatility of R and by providing various novel functionalities for performing data preparation, visualization, and comparison. In addition, thanks to its interoperability with a plethora of existing R packages in e.g., Bioconductor, CRAN, and GitHub, KPM-R allows carrying out the initial preparation of the datasets and to meaningfully interpret the extracted subnetworks. To demonstrate the package's potential, KPM-R was applied to bulk RNA-Seq data of nasopharyngeal swabs from SARS-CoV-2 infected individuals, and on single cell RNA-Seq data of aging mice tissue from the Tabula Muris Senis atlas. values, DNA methylation signals or single nucleotide variants which can come from primary data or can be obtained through online databases and repositories, while biological interaction networks can be derived from protein-protein-interaction databases or gene regulatory networks. The R statistical framework with its rich package ecosystem (R Core Team, 2020) and its Bioconductor repository allows for an easy retrieval for this kind of data. Moreover, thousands of R packages exist in CRAN, Bioconductor, and GitHub, which provide functionalities in the acquisition, preparation, normalization, and visualization of the datasets. While several tools have been proposed for pathway enrichment [bib_ref] On the Performance of De Novo Pathway Enrichment, Batra [/bib_ref] , the integration of de novo pathway enrichment in end-toend OMICS analysis workflows in R is to our knowledge currently limited to . De novo pathway enrichment methods generally vary in their performance, depending on the dataset and problem setting selected [bib_ref] On the Performance of De Novo Pathway Enrichment, Batra [/bib_ref]. A complementary pathway enrichment tool, KeyPathwayMiner (KPM) , is currently only available as a Cytoscape app, a standalone Java application or via a web server, making its integration into R-based workflows challenging for typical R users who often lack experience with lower level programming languages and the use of the command line interface. Moreover, the preparation of the suitable input files for KPM from primary data, e.g., a network in SIF format and an indicator matrix of features vs samples, is not trivial for most users. To close this gap, we have implemented an R package KeyPathwayMineR (KPM-R) with extended features and improved usability. To make the integration of KPM-R straightforward, we implemented a multitude of convenience functions. For example, the package's methods facilitate the retrieval of biological interaction networks from BioGRID [bib_ref] The BioGRID Interaction Database: 2019 Update, Oughtred [/bib_ref] for various organisms. In our vignette we show how OMICS data from repositories like GEO [bib_ref] Geoquery: a Bridge between the Gene Expression Omnibus (Geo) and Bioconductor, Davis [/bib_ref] or TCGA [bib_ref] Tcgabiolinks: an R/bioconductor Package for Integrative Analysis of Tcga Data, Colaprico [/bib_ref] can easily be downloaded and processed internally into a suitable input format for KPM-R. We further added support for the analysis of single cell RNA sequencing data where users can provide a single-cell RNA-seq object and can pick the comparison to be made. KPM-R allows switching between a local and remote execution depending on the user's needs. If the user lacks sufficient computing power, the analysis can be run through the provided RESTful service on the KPM web server. The user can also conveniently define a parameter range to simplify the otherwise tedious hyperparameter optimization. KPM-R visualizes the results across hyperparameters to allow determining the most appropriate choice. For the visualization of the extracted de novo pathways, we implemented an interactive web app using the R shiny framework. This allows users to browse, visualize and export the sub-networks as an image or machine-readable text files. In contrast to the existing web application (https:// keypathwayminer.compbio.sdu.dk/keypathwayminer/), the shiny app can also be used to visualize results that were created offline and serves as a template for users that want to embed KPM-R in more complex shiny apps for data analysis. The vignette also offers guidance of further downstream analysis of the selected pathways which includes gene ontology and gene set enrichment analysis. To demonstrate the benefits of KPM-R for assembling R data analysis workflows, we used a large dataset of nasopharyngeal swabs from SARS-CoV-2 infected individuals and negative control cases (GSE152075) [bib_ref] In Vivo antiviral Host Transcriptional Response to Sars-Cov-2 by Viral Load, Sex,..., Lieberman [/bib_ref]. Furthermore, we investigated celltype-specific subnetworks in primary mouse tissue from single cell RNA-seq data from the Tabula Muris Senis Atlas [bib_ref] A Single-Cell Transcriptomic Atlas Characterizes Ageing Tissues in the Mouse, Almanzar [/bib_ref]. In both application cases, we show how the package assists in processing raw data, finding de novo pathways relevant to the studied diseases and conducting downstream analyses that offer a more in-depth understanding of the disease mechanisms. # Materials and methods ## Strategies for network extraction The objective of KPM is to extract maximally connected subnetworks that are enriched in differentially active nodes which can represent e.g., differentially expressed genes. The user can define which nodes are considered active in each case or sample by supplying an indicator matrix. The rows of the matrix describe the nodes (typically genes or proteins) and the columns the samples (see Subsection 2.3.1 for more information on indicator matrices) . The task of finding the maximal subnetwork can be defined as a graphtheoretical problem: Let graph G (V, E) represent an undirected biological network that consists of a set V representing biological entities and a set E containing the interactions between them. Further, we have one or, in case of multi-omics data, multiple indicator matrices A 1 , . . ., A p , e.g., an indicator matrix from a proteomics experiment and another one from a transcriptomics study. The indicator matrices consist of n features (e.g., genes) and m samples (e.g., patients). These matrices will be combined to a single indicator matrix A n,m . To combine the indicator matrices, the biological entities should be identical for all matrices and the number of samples should be equal. Additionally, we have a mapping Z: V → {0,1} m from each node v ∈ V to its corresponding row in the matrix A. The objective is to extract a subnetwork G′ (V′, E′) ⊂ G such that G′ is connected and the set of nodes V′ satisfy the constraints of the INES or the GloNE strategy . ## Individual node exceptions strategy The INEs strategy uses two parameters K and L to constrain how maximally connected subnetworks enriched with differentially active nodes are extracted. The L parameter describes the maximal number of allowed case exceptions for a particular node in the extracted network (i.e., in an active node, the number of patients/samples in which the corresponding entity (e.g., gene or protein) is not differentially regulated must be less or equal to L). K is the number of allowed inactive nodes in the extracted subnetwork (i.e., they exceed the number of allowed case exceptions). Intuitively, L regulates which genes are considered relevant whereas K allows KPM to combine smaller solutions into larger ones using up to K genes as connectors. Due to their central role in the subnetwork such nodes may be important for a pathway even if they are not differentially expressed or regulated themselves . Formally, this can be described in the following way: Given a biological network G (V, E) , an indicator matrix A n,m , K node exceptions, L case exceptions and s being a sample from the indicator matrix extract maximally connected subnetworks G′ (V′, E′) for which following formula evaluates to true for all nodes v ∈ V′ but K exceptions: [formula] m − m s 1 A v,s ⎛ ⎝ ⎞ ⎠ ≤ L. [/formula] . ## Global node exceptions strategy The GloNE strategy only considers the L parameter. L describes the number of allowed case exceptions over all nodes of the extracted subnetwork G′. This means in an extracted subnetwork, the number of patients/samples that are not differentially regulated over all nodes must be less or equal to L. In contrast to INES, we do no longer consider each gene individually but have a global budget of exceptions that can be spent for maximizing a solution. Nodes which are active in most cases will thus be cheaper to add to an existing solution than nodes which are mostly not active. The strategy can be formally defined as follows : Given a biological network G (V, E), an indicator matrix A n,m , L case exceptions and s being a sample from the indicator matrix extract maximally connected subnetworks G′ (V′, E′) for which the following formula evaluates to true: [formula] |V′| p m − v∈V′ m s 1 A v,s ⎛ ⎝ ⎞ ⎠ ≤ L. [/formula] ## Algorithms for network extraction INES and GloNE are both computationally hard problems. Three types of algorithms have been implemented to make the computation of the solutions as efficient as possible. ## Fixed parameter tractable To solve the INES problem for small K values a fixed-parameter tractability approach for extracting exact solutions can be used. The approach applies an exact branch and bound algorithm to extract optimal subnetworks. If for a given x, the algorithm has a partial solution which already has K − x exception nodes, the algorithm computes the upper bound in the following steps: First it determines all possible new exception nodes that are reachable from the current subgraph in x steps and then considers the n nodes with the highest weights and adds those together. Once the upper bound is determined, the algorithm uses the two bounds in an exhaustive search to find the optimal solution . ## Greedy The Greedy approach offers an efficient way to tackle large problem instances like dense networks. The algorithms starts by adding a seed node v to an empty partial solution S {} and then iteratively adds the adjacent node u to the partial solution that maximizes the following scoring function: [formula] f(S, u) r∈(V(S))∪{u} p i 1 m s 1 A r,s i ; [/formula] The process is repeated until no more nodes are left which could be added to gain a valid solution . ## Ant colony optimization The third option is a heuristic approach based on Ant Colony Optimization called Max-Min Ant System (MMAS). Given enough time this algorithm can provide an improved solution compared to the Greedy approach. The ACO algorithm starts from a seed node v from where you can imagine multiple ants searching for valid solutions. The ants add the next valid node based on a probability proportional to η(u) αp τ(u) β . η(u) is a heuristic value of node u which is proportional to the number of active cases and τ(u) is the current pheromone level of node u, which is proportional to the amount of ants which have previously used this vertex to create a valid solution, while the parameters α and β control the importance of η(u) and τ(u). When there is no valid node left for any ant to add, the pheromone levels are updated. First the pheromone values of all edges are decreased by an evaporation factor. Then the best solution is extracted and the pheromone levels for the nodes within this network are increased. The whole procedure is repeated until it converges to one solution . ## Implementation KPM-R, is based on two previously developed libraries: 1) the KPM-Standalone library, and 2) the KPM-Web library . The two KPM libraries allow the user to switch between a local and remote execution within the R package. The local version executes the KPM standalone jar, which is included when installing the KPM-R from GitHub; the remote version utilizes the KPM webserver to execute the method. The communication between the standalone and R application was established using the rJava package, a low-level R to Java interface. In the remote execution, the RESTful API from the KPM-Web module was utilized. To access the Web-API, KPM-R uses the RCurl package to create HTTP requests (Temple Lang, 2020) and the rjson package to convert R objects into JSON ones (Couture-Beil, 2018). ## Input data KPM-R requires two types of input data. The first type is a biological network from which the pathways should be extracted. Some examples of networks that can be used are HPRD (Keshava, STRING [bib_ref] String V11: Protein-Protein Association Networks with Increased Coverage, Supporting Functional Discovery in..., Szklarczyk [/bib_ref] , or BioGRID [bib_ref] The BioGRID Interaction Database: 2019 Update, Oughtred [/bib_ref]. These networks can either be downloaded with the help of R packages or manually from the corresponding website. A plethora of biological networks can be found and downloaded from the Network Data Exchange [bib_ref] NDEx: A Community Resource for Sharing and Publishing of Biological Networks, Pillich [/bib_ref] using the NDExR package. For an example on how to use NDExR and prepare the downloaded networks for KPM-R, see our vignette. KPM-R accepts a biological network in three ways: as the file path to where the network is located (in SIF format), as an igraph object, or as a network from the KPM web server, which is exclusively for remote execution and allows for selecting one of the web server's multiple networks. Every network on the server is assigned a unique id, which can be observed using get_networks(). Once the desired ID is determined, the user can specify the graph ID with the options function, for instance, like this kpm_options(graph_id 2). The second type of input is one or multiple indicator matrices derived from OMICS datasets. The rows of a matrix represent the biological entities (e.g., genes), and the columns the cases/ samples. In the case of a differential expression experiment a column would indicate if the genes of a certain sample (e.g., a patient) are differentially expressed compared to the control samples (e.g., the healthy individuals). An entry of "1" would indicate differential expression for a gene between a sample and the reference. All other entries have to be "0"s. There are several possibilities for a user to produce such an indicator matrix. For instance, in a differential expression analysis comparing two groups (e.g., control vs. diseased), we can either compute a group-wise statistic (e.g., t-test) that results in an indicator matrix with one column where each entry describes whether a gene is differentially expressed or not. However, to leverage the potential of KPM we are more interested in sample-wise statistics where one value is computed for each gene and sample pair. To compute such an indicator matrix, we propose to compute the parameters (mean μ c and standard deviation σ c ) of a normal distribution based on the control samples. For each case sample s and gene with expression x s , we can then compute a z-score using the formula x s −μ c σ c . Intuitively, this will yield a z-score that indicates how many standard distributions an expression value is away from the mean of the control group. The resulting matrix of z-scores can then be binarized using a user-defined threshold on the absolute values of the matrix. For details we refer to 2.4. After applying the selected statistic, the user can decide which genes are differentially expressed based on the p-value and log fold change (or z-score) and whether to consider up-regulated or down-regulated genes, or both. In our vignette we give two examples on how to construct an indicator matrix. The first column of the matrix has to contain the IDs of the biological entities. The ID type of the biological entities should be equivalent to the ID type of the entities in the network. The matrices should be processed so that they do not have a header in the first row. The matrix can be passed to KPM-R as a data frame and, in the case of multiple matrices, as a list of data frames. ## Execution parameters Once the input data have been prepared, the user can set the execution parameters using the kpm_options() function. The general way to change an option is kpm_options [value]), where the key stands for the parameter to be changed and the value for the parameter value to be set. All parameters are case sensitive. The user can also provide multiple key-value pairs separated by a comma in one command. In total, 31 parameters can be used to adjust the execution settings to the user's preferences. For most of the parameters, a default value is defined, which allows the execution of KPM-R with the configuration of just two parameters, K and L. The most important parameters of KPM-R can be found in [fig_ref] TABLE 1 |: KPM run options and their description [/fig_ref]. A complete list of all options and their default values can be retrieved when running the command ?kpm_options(). ## Data processing Several convenient functions were implemented to make the user's data processing workflow as easy as possible. One of them is the compute_z_score() function, which computes the genes' z-scores in all case samples while using as background the control samples. The function receives a count matrix as input and returns a z-score matrix. The z-score of a gene i in a sample j is computed in the following way: Another convenient function is the to_indicator_matrix() function. This function converts a p-value matrix, describing the significance of the biological entities, into an indicator matrix. The function receives two parameters as input, which are the p-value matrix and the threshold for setting active entities. The import_graph() function allows the user to convert their graph file into an iGraph object, which is the input format required by the package. The user can choose from a variety of graph file formats, such as sif, gml, graphml, xlsx and documents with user-defined delimiters. Furthermore, the user can utilize the export_graph() function to export pathways computed by the package. Given a pathway, the user can export the network in one of the following formats: sif, gml, graphml, xlsx, csv, igraph object or using a customer delimiter. The user can also extract only the nodes of the pathway by using the export_nodes() function. ## Input of single cell rna-seq data The function sc_to_indicatormatrix() allows to generate an indicator matrix based on differential expression from single cell input data. The differential expression detection is performed by a two-part generalized linear model implemented in the MAST package which allows to address the additional complexity of scRNA-seq data and also adjustment for covariates. The user has to provide single cell RNA-seq data which is normalized but not transformed, yet. The data is accepted in form of a Seurat [bib_ref] Spatial Reconstruction of Single-Cell Gene Expression Data, Satija [/bib_ref] , SingleCellExperiment [bib_ref] Orchestrating Single-Cell Analysis with Bioconductor, Amezquita [/bib_ref] or SinglCellAssayobject. In case of a Seurat Object the data should be in the Assay named "RNA". For SingleCellExperiment objects the first assay is considered. The input data is log2 transformed and a hurdle model is fitted using MAST's zlm function. This generalized linear framework can be used to jointly estimate variation from biological and technical sources, as well as the effects of interest. The function controls by [bib_ref] Controlling the False Discovery Rate: A Practical and Powerful Approach to Multiple..., Benjamini [/bib_ref] and data is filtered by p-value and fold change which can be chosen by the user. As the method takes into account that multiple samples can be derived from the same individual, type 1 error rate is reduced and compared to a pseudo bulk approach, a type 2 error inflation is avoided. ). Subsequently, a differential expression analysis was carried out in which the z-scores of the genes in the case samples were calculated using the z-score function (Subsection 2.4). Three different cutoffs were used to determine the best-suited z-score for the dataset. For every applied cutoff, the average number of differentially expressed genes over all samples was computed. From these observations, an indicator matrix was created for which the z-score cutoff of 2 was taken. Together with the human BioGRID PPI network an INES run with the Greedy algorithm was performed using L parameter values between 20 and 220 with a step size of 20 and K parameter values between 2 and 20 with a step size of 2. An enrichment analysis was performed with the profile_pathway function using the pathway with the configuration L 220 and K 20. Significant results with the highest intersection size were further manually inspected. ## Use-case data ## Tabula muris senis data Processed scRNA-seq data was retrieved from Figshare https:// doi.org/10.608 4/m9.figshare.12 827 615.v3 by downloading the rds_by_tissue.14.zip file. The droplet. normalized.Limb_Muscle.rds file was then selected and was filtered for "mesenchmymal stem cells" and "mesenchmymal satellite stem cells". Mice aged 1 and 3 months were considered as young control cases while mice aged 18, 21, 24, and 30 months were treated as old. Differential expression between young and old mice was performed based on a linear mixed effect model from the MAST package while controlling for sex and number of genes per cell. Each mouse of the old age group was treated as a case and compared against all young mice, resulting in 12 in comparisons. Genes below a Benjamini-Hochberg FDR corrected p-value of 0.05 were treated as differently expressed and considered as active in the indicator matrix. The Mus musculus BioGRID was chosen as a biological network and filtered for genes which were expressed in the mouse tissue. Given the input data, a grid run with L values between 1 and 6 and K values from 0 to 10 with the INES strategy and the greedy algorithm was performed. # Results ## Workflow The lack of a user-friendly solution for de novo enrichment in the R ecosystem motivated us to develop KPM-R. The typical Parameter Description execution Defines the execution type of KPM-R, which can be run either "Local" via RestfulAPI or "Remote" via standalone jar. Default value: "Local". strategy Can be either "INES" or "GLONE". If the GloNE strategy is selected, the user does not need to set the K parameter. Default value: "GLONE". algorithm The algorithm that should be used to extract the pathways. It can be set to "Greedy", "ACO" or "Optimal". Default value: "Greedy". use_range_k Boolean parameter that describes whether parameter K should be ranged or not (see below). Default value: FALSE. k_min, k_max, k_step Numeric parameters that control the number of node exceptions allowed in a solution. If the use_range_k parameter is set to false, only k_min must be defined. Otherwise, a range must be defined with k_min and k_max defining the lower and upper boundary respectively and k_step describing the incrementation from one iteration to the next. For example, setting k_min 4, k_max 8 and k_step 2 would mean that KPM will be executed with K 4, K 6 and K 8. When using multiple datasets, the user must specify a logical formula to combine these. The link_type parameter's accepted values are "OR", "AND", or a custom formula. Default value: "OR". graph_id ID of the network on the web server, which should be used in a remote run negative_nodes Character vector contains biological entities that should be considered as inactive positive_nodes Character vector contains biological entities that should be considered as active Frontiers in Genetics | www.frontiersin.org January 2022 | Volume 12 | Article 812853 workflow when using KPM-R can be divided into three steps, also depicted in : ## Data preparation One or multiple OMICS datasets and a biological network are loaded into R. The biological network has to be either saved as a file in one of the supported graph formats (see 2.4) or has to come as an igraph object. Using KPM-R's internal function retrieve_biogrid() various biological networks from BioGRID can easily be loaded in the proper format. The user can select the gene identifier and the organism which match the OMICS data. OMICS datasets must be transformed into indicator matrices if they are not already in this form. Each matrix entry is either a '1' indicating an active case in a node or '0' otherwise. For example in a gene expression dataset a '1' would represents a differentially expressed gene or in bisulfite sequencing data a differentially methylated promotor of a gene. Optionally the user can also incorporate previous knowledge and provide positive and negative lists defining genes which are always considered active or that are ignored. For the generation of indicator matrices KPM-R comes with two convenient functions. The to_indicator_matrix() function converts a p-value matrix, describing the significance of the biological entities, into an indicator matrix. The sc_to_indicator_matrix() functions allows to take input of single cell RNA-seq data in various common object formats. ## Execution of kpm-r Once the datasets are prepared, the run's execution parameters must be set. Most importantly, the user has to decide to either use the Individual Node Exceptions (INES) strategy, or the Global Node Exceptions (GloNE) strategy for the discovery of meaningful pathways. Both strategies are computationally hard problems and subnetwork extraction can either be based on a greedy approach, a heuristic based on Ant Colony Optimization or, for INES, even by calculation of the exact solution for small K values using a fixed-parameter tractable algorithm. The parameters for a run can be set with the kpm_options() function, where the user has the possibility to define ranged parameter values instead of unique ones. The optimal values for K and L depend on the dataset, therefore the function allows several iterations with different configurations of K and L values to be performed in one run. Generally, larger K values result in larger networks. When choosing the parameter the user has to be aware of the underlying hypothesis of his experiment. For instance, the up to K exception nodes that correspond to genes that are not differentially expressed may still be disease-relevant due to mutations. Alternatively, they may pose promising drug targets as they are of central importance to diseaseassociated genes. Choosing large K values, however, increasingly leads to the incorporation of hub nodes which connect to distant parts of the network and thus different pathways which may not be functionally related. For the choice of the L parameter, the user should look at the ratio of the number of samples and a possible L value. For example, an L value of 10 in a study with 20 cases would mean that for a given node, at most 50% of the cases may not be differentially expressed. Users need to select a suitable value here which fits their expectation but they can also perform a grid search to conveniently explore different L values. The parameters of a run can be globally set and, subsequently, the kpm() function can be used to execute the program given a biological network and a list of indicator matrices as input. Once the execution of the program is completed, the results are saved in a unified manner, and an export function allows the user to easily extract the pathways as igraph objects or in several graph formats. ## Pathway visualization The result object obtained from the run can then be used to visualize, browse and save pathways with an interactive web app which was developed using Shinyand visNetwork [bib_ref] visNetwork: Network Visualization Using 'vis, Almende [/bib_ref]. The function visualize_results() allows the user to browse through the pathways and also to save them as an image or text file. A node in a pathway can be in two states, either in an exception state where the number of inactive cases for this node is above the L value or in a significant state in which the number of inactive cases is at most L. Nodes in an exception state are symbolized by an orange square and nodes in a significant state are represented by a blue circle. This does not apply to the GLONE strategy, which does not contain exception nodes and therefore only contains circles. If the nodes in the subnetworks are genes, the user can click on them to be forwarded to the corresponding gene entry on the NCBI website [bib_ref] The National center for Biotechnology Information, Benson [/bib_ref]. In , the interface and the single components of the Shiny app are presented and described. Using the function pathway_comparison_plots(), two plots are generated: one comparing the top pathways and one comparing the union networks (all pathways of a configuration merged) of every parameter-configuration (see for an example). Specifically, the plots compare all parameter configurations by plotting the average active cases per node (e.g., the sum of differentially expressed cases for a certain gene) against the number of nodes in a pathway. These plots serve as an aid to users to select the best pathways for in detail exploration and further downstream analysis. When the user has found an interesting pathway, the shiny app allows to easily download this network in SIF format, simply by clicking the button "Export edges (SIF)". Finally, the user can also directly perform downstream analysis with the profile_network() function and easily visualize its result with gprofiler2's plot functions. ## Use cases Two application cases were selected to evaluate the usability and features of KPM-R. The use-cases aimed to demonstrate the benefits of conducting an analysis with KPM-R, the integration of KPM-R with Bioconductor, and the power of KPM-R in finding meaningful results. The majority of the analysis steps, from the data acquisition to the further downstream analysis of the results, were conducted within the R framework. ## Sars-cov-2 infection The COVID-19 pandemic has confronted our society with major challenges. For this reason, research into understanding and combating the infectious agent, SARS-CoV-2, is important. The amount of publicly available data generated in context to SARS-CoV-2 is continuously increasing and can be used for further in depth analysis. Here, we used a large bulk RNA sequencing dataset of nasopharyngeal swabs from 430 SARS-FIGURE 3 | Pathway comparison plots can be utilized to find the optimal pathway in the extracted solution. The shown pathways were extracted from the analyzed GEO SARS-CoV-2 dataset and used to limit the further exploration to configurations with at least 100 on average differentially expressed genes per case. Frontiers in Genetics | www.frontiersin.org January 2022 | Volume 12 | Article 812853 8 CoV-2 infected individuals and 54 negative control cases. The raw counts were directly downloaded into R using the GEOquery package [bib_ref] Geoquery: a Bridge between the Gene Expression Omnibus (Geo) and Bioconductor, Davis [/bib_ref] and subsequently normalized using edgeR [bib_ref] Differential Expression Analysis of Multifactor Rna-Seq Experiments with Respect to Biological Variation, Mccarthy [/bib_ref]. Mean and standard deviation was calculated for every gene using the control group's gene counts as reference. Genes with a z-scores below −2 and above 2 were considered as differentially expressed. With the generated indicator matrix and the BioGRID as a biological network, KPM-R was executed in a grid run for L values between 20 and 220 with step size 20 and K values between 2 and 20 with step size 2, using the INES strategy and the Greedy algorithm. Intuitively, this means that the genes in the extracted solutions will be in at most 51% (220/430) of the studies inactive and that an extracted solution will have at most 20 exception genes. For finding the most promising pathways a comparison plot was generated . Networks with on average more than 100 differential expressed cases were examined. Exploring these results we find a network with many known and recently described key players of Covid-19 . Looking at the leaf nodes, we see many genes which are an important part of human immune response like interferon induced genes IFIT1/2 and IFI16 or chemokine ligands like CCL5/9/10/11. Inspecting the central nodes we find the antiviral kinase EIF2AK2, also an important actor in the innate immune response [bib_ref] Rna-activated Protein Kinase Differentially Modulates Innate Immune Response Mediated by Supraphysiological Concentrations..., Ishaq [/bib_ref]. Its direct neighbor TP53 is known to FIGURE 4 | SARS-CoV-2 network from the configuration L 220 and K 20. Many genes of the network are known to be important players in the human immune response. Exception nodes are visualized as orange squares and significant nodes as blue circles. facilitate EIF2AK2's expression and also acts as host antiviral factor by itself. Recently, SARS-CoV-2's papain-like protease of the nonstructural protein 3 was shown to downregulate TP53 at the protein level by manipulations of it's ubiqutiniation and thereby facilitating viral replication [bib_ref] p53 Down-Regulates SARS Coronavirus Replication and Is Targeted by the SARS-Unique Domain..., Ma-Lauer [/bib_ref]. Although TP53 is an exception node and hence does not show differential abundance on the transcript level, SARS-CoV-2's induced TP53 degradation might contribute to EIF2AK2's downregulation and consequently further weaken the anti viral defense. EIF2AK2 is also linked to STAT3, another important regulator of the immune response. In many Covid-19 patients STAT3 is hyperactivated which is associated with cytokine release syndrome and acute lung injury [bib_ref] An Aberrant Stat Pathway Is central to Covid-19, Matsuyama [/bib_ref]. Moreover, STAT3 is described to inhibit PKR's activity and hence its overexpression potentially leads to another weakening of the viral defense mechanisms. STAT3 is an exception node and is only differentially expressed in 6% of the patients, therefore this interaction might only be relevant for a subtype of patients. Unfortunately, we lack the metadata to see if the differential STAT3 expression could be linked to features like the severity of the infection. STAT3's function could also be affected on the protein level by adjacent nodes. It's a strength of KPM that the results can include genes which might not be differentially expressed on the transcript level but still be part of a biological cascade. The network also shows an interaction between STAT3 and its adjacent node CCR5 which has STAT3 binding sites in its promoter [bib_ref] Stat3/ccr5 Signal Regulation Mitigates Renal Ischemia-Reperfusion injury, Yoo [/bib_ref]. CCR5 has already been part of a initial clinical trials which showed that its inhibition can decrease inflammatory cytokines in Covid-19 patients [bib_ref] Ccr5 Inhibition in Critical Covid-19 Patients Decreases Inflammatory Cytokines, Increases Cd8 T-Cells,..., Patterson [/bib_ref]. To make sure that we were not exploring a previously described pathway, we performed functional enrichment analysis with our integrated pathway enrichment function. Here we checked for enrichment within KEGG, Reactome and WikiPathways and filtered for the significant hits with the highest intersection size (number of genes of our extracted network found within a certain pathway) . We found that various genes of our network are also part of known pathways in viral infections including SARS-CoV-2. However, at most 30% of the genes detected by KPM were part of one of these networks. The highest overlap was found with the terms "Immune System" and "Cytokine Signaling in Immune system" where respectively 55 and 70% of the genes in our network could be found. Still our network contains around 30% genes which were not described in these pathways, indicating that we could indeed have found a pathway which was not described previously. We leave further exploration and interpretation of the data to the experts in the field. We hope that examination of the networks generated by KPM-R might even offer potential clues for the experts on how to infer with one of these processes. ## Aging tissue from tabula muris senis Muscle tissue in mammals has various functions including mobility, body temperature regulation, energy storage and support of soft tissue. However, muscle mass and function decline in mammals with age, a condition named sarcopenia. Understandably, the loss of muscle mass and function is associated with numerous morbidities and mortalities and sarcopenia has a great impact on healthcare costs [bib_ref] Sarcopenia: Aging-Related Loss of Muscle Mass and Function, Larsson [/bib_ref]. Rodents and humans suffer from a decline in muscle tissue with aging in a comparable way which makes them a good model organism for the study of muscle atrophy [bib_ref] Rodent Model of Muscular Atrophy for Sarcopenia Study, Baek [/bib_ref]. The Tabula Muris Senis, or 'Mouse Ageing Cell Atlas' is a great resource for transcriptomic data of the aging mouse tissue [bib_ref] A Single-Cell Transcriptomic Atlas Characterizes Ageing Tissues in the Mouse, Almanzar [/bib_ref]. The atlas consists of single-cell RNA sequencing data of more than 350,000 cells from male and female mice tissues belonging to six age groups, ranging from 1 month to 30 months. Mice at the age of 1 month can be compared to humans at a early childhood, while 30 months old mice are the equivalent of a human centenarian [bib_ref] A Single-Cell Transcriptomic Atlas Characterizes Ageing Tissues in the Mouse, Almanzar [/bib_ref]. We aimed to use KPM-R to identify differentially regulated pathways in the aging tissue. Therefore, we used single cell data from the limb muscle and focused on the mesenchymal stem (MSC) and satellite cells. In the muscle tissue, MSCs can give rise to satellite cells. While these cells which are named after their satellite position on the myofibre, differentiate into myoblasts and are responsible for muscle repair and growth. Mice age 1 and 3 months were treated as young control cases and were compared to the remaining time points. We used our internal functions based on MAST package's linear mixed effect model for finding differentially expressed genes between the young and old mice. Together with the Mus musculus interaction network from BioGRID an INES run with L values between 0 and 6 and K values between 0 and 10 was performed. Inspecting the MSC results we could extract a small network where each of the genes is differentially expressed in all aging mice [fig_ref] FIGURE 6 |: Extracted networks from single cell data of murina limb muscle [/fig_ref]. It consists of extracellular matrix proteins including members of the collagen family like Col1a1/2, fibrillin-1 and proteins potentially involved in the processing of these proteins like the metalloprotese Adamts2. Collagen is the most abundant protein in mammals and depending on the tissue it consists of a variable mixture of different collagen proteins. In muscle tissue it serves as the major component of the endomysium which ensheats each muscle fiber. During aging the skeletal muscle's connective tissue compartment is known to show significant changes [bib_ref] Age-related Changes in Collagen Gene Expression in the Muscles of Mdx Dystrophic..., Goldspink [/bib_ref] [bib_ref] Human Aging, Muscle Mass, and Fiber Type Composition, Evans [/bib_ref]. The computed network can be seen as a proof of KPM-R's ability to find biological meaningful interactions among the single cell dataset. Taking a further look at the networks of the satellite cells we see various networks which include a highly connected networks of ribosomal proteins [fig_ref] FIGURE 6 |: Extracted networks from single cell data of murina limb muscle [/fig_ref]. It was recently shown that dozens of proteins involved in the ribosome biogenesis are down regulated with age which is consistent with age related decline in protein synthesis [bib_ref] Multifaceted Deregulation of Gene Expression and Protein Synthesis with Age, Anisimova [/bib_ref]. Enrichment analysis showed that this network is part of already annotated pathways. However, we found these networks of ribosomal proteins also being connected to various other proteins which might make them worth exploring for the specialists in the field. # Conclusion KeyPathwayMineR is an established tool in the field of de novo pathway enrichment and was so far available as a Java stand alone tool, a web-server version and a Cytoscape plugin. Here we have created a package of KPM in the R programming environment which has a rich community of biological scientist who appreciate easily understandable workflows. Due to KPM-R's interoperability with a plethora of existing R packages, which can be found on Bioconductor, CRAN, or GitHub, KPM-R allows access to a large number of datasets, networks, data processing and down-stream analysis methods which can now be conveniently integrated into fully reproducible analysis workflows. We demonstrate the packages abilities by applying KPM-R on a large bulk RNA-seq dataset of nasopharyngeal swabs from SARS-CoV-2 infected individuals. The data of this study was downloaded and prepared in R with well known packages like GEOquery and edgeR. After performing a grid run with KPM-R, the pathway-comparison-plots function allowed us to easily find the most promising networks. The inspected SARS-CoV-2 network contained many already described key player in Covid-19 disease like STAT5, EIF2AK2 or various chemokines. We also found TP53 as a common interaction partner in the network and a transcription factor of its neighbor node EIF2AK2. Although TP53 did not show differential abundance at the transcript level it's known to be downregulated by SARS-CoV-2 at the protein level. Hence we FIGURE 7 | Network from single cell data of satellite stem cells from the murine limb muscle with the configuration K 6 and L 4. Exception nodes are visualized as orange squares and significant nodes as blue circles. show that exception nodes can still be meaningful interaction partners and KPM-R coupled with domain knowledge allows to find potentially interesting pathways. During the last years, single cell RNA sequencing became increasingly popular and is slowly replacing bulk RNA sequencing as the major method to study transcript abundances. In KPM-R we also implemented functions which make it easy to work with single cell RNA sequencing data. Using single cell data of the Tabula Muris Senis Atlas, we show that KPM-R can find potentially interesting pathways in the aging murine muscle tissue. The mesenchymal stem cells showed differentially expressed networks containing extracellular matrix proteins and in satellite cells we saw densely connected nodes of ribosomal proteins. Both remodelling of the skeletal muscle's connective tissue and ribosome biogenesis are processes already previously linked to aging. We leave these networks to be further explored by the experts in the field. In conclusion, KPM-R extends the features and usability of existing versions of KPM by leveraging the power, flexibility and versatility of R, thereby providing R users with various functionalities for performing data preparation, de novo pathway enrichment and visualization. # Data availability statement Publicly available datasets were analyzed in this study. This data can be found here: The datasets for this study were downloaded from the Gene Expression Omnibus (GSE152075) and from figshare (https://doi.org/10.6084/m9. figshare.12827615.v3). # Author contributions KM and MLa implemented the package and wrote the manuscript. MLi and JB supervised the work and revisited the manuscript. All authors read and approved the manuscript. # Funding MLa was supported by the Hanns-Seidel-Stiftung. JB is grateful for support from H2020 grant RepoTrial (nr. 777111) and his VILLUM Young Investigator grant (nr. 13154). [fig] FIGURE 6 |: Extracted networks from single cell data of murina limb muscle. (A) Network with the configuration L 0 and K 0 based on mesenchymal stem cells from the limb muscle. The genes consist mainly of extracellular matrix proteins and are differentially expressed in all old mice. (B) Network of mesenchymal satellite stem cells with K 0 and L 4. With the exception Tpt1 and Gnbl2l1, all nodes are ribosomal proteins.Frontiers in Genetics | www.frontiersin.org January 2022 | Volume 12 | Article 812853 [/fig] [table] TABLE 1 |: KPM run options and their description. [/table]

Recurrent nonbacterial thrombotic endocarditis and stroke on anticoagulation We present a rare case of recurrent nonbacterial thrombotic endocarditis (NBTE) and stroke despite anticoagulation. A 48-year-old man with history of antiphospholipid syndrome, prior nonbacterial aortic valve endocarditis status post valve replacement and prior stroke was found to have acute ischemic stroke while on apixaban and nonbacterial thrombotic endocarditis of mitral valve. This was initially managed conservatively with therapeutic dose of enoxaparin, but the patient later underwent mitral valve replacement. Unfortunately, the patient later passed away with hemorrhagic stroke while on enoxaparin.ARTICLE HISTORY # Introduction Nonbacterial thrombotic endocarditis (NBTE), usually discovered postmortem, is a rare noninfectious cause of endocarditis and is characterized by fibrinoplatelet deposition on heart valves. Its prevalence ranges between 0.3% and 9.3%. NBTE is most commonly associated with primary antiphospholipid syndrome (APS), malignancy, hypercoagulable states, and systemic lupus erythematosus (SLE). NBTE in the setting of APS or SLE is also called Libman-Sacks endocarditis. We present a rare case of recurrent NBTE characterized by mitral valve endocarditis complicated by stroke despite therapeutic anticoagulation in the setting of primary APS and previous aortic valve endocarditis status post aortic valve replacement. ## Case description The patient is a 48-year-old man brought to the Emergency Department in March 2019 from a nursing home with new right-sided facial droop. The patient had baseline cognitive impairment with left-sided weakness and visual field deficit from previous strokes. He was last known well the day earlier before he went to bed the previous night. He did not have any new arm or leg weakness. His past medical history was significant for antiphospholipid syndrome, NBTE of his aortic valve status post aortic valve replacement with Trifecta bioprosthetic valve 5 years back, occipital stroke with residual inferior visual field deficits, and multiple prior pulmonary embolisms and deep vein thromboses with inferior vena cava filter placement. He had previously failed warfarin and rivaroxaban and was on apixaban 10 mg two times daily. He had failed warfarin at another hospital as his international normalized ratio was never therapeutic from suspected Protein C or Protein S deficiency. Therefore, he was placed on rivaroxaban in September 2014. He was changed to apixaban during a hospitalization for a stroke in June of 2015 while on rivaroxaban. He was on apixaban 5 mg two times daily with last dose an hour prior to presentation. On examination, his temperature was 97.2°F and he was noted to have bilateral inferior visual field deficits from previous stroke. He performed one task correctly, answered one question correctly, had flattened nasolabial fold with asymmetry while smiling, and loss of fluency with National Institute of Health Stroke Scale of 4. Rest of the examination was unremarkable. He was deemed not a candidate for recombinant tissue plasminogen activator as he had received apixaban an hour prior to presentation. A stat computed tomography of head showed encephalomalacia along bilateral superior and posterior parietal lobes with age indeterminate areas of low attenuation along the superior left frontal lobe extending to the cortex which were concerning for infarcts. Diffusion-weighted magnetic resonance imaging of the brain showed acute versus subacute punctate foci of hyperintensity within the left medial occipital lobe and bilateral superior parietal lobules along with remote infarcts involving the superior middle frontal gyri, left frontal opercular regions and extending into bilateral occipital regions. Magnetic resonance angiogram of the head and neck did not show any hemodynamically significant stenoses in intracranial circulation, cervical carotids, and vertebral arteries. Electrocardiogram showed sinus rhythm with first-degree AV block. He was started on aspirin 81 mg daily and atorvastatin 40 mg daily. Hematology was consulted who recommended lifelong anticoagulation with enoxaparin 104.3 mg two times daily based on his weight. Given history of aortic valve endocarditis and replacement and concern for embolic phenomenon, transesophageal echocardiogram (TEE) was performed. It showed a well-functioning stented bioprosthetic aortic valve with trace aortic insufficiency and no evidence of thrombus or vegetation. However, a 0.7 × 1.1 cm mass noted on the tip of anterior mitral valve leaflet and 0.7 × 0.9 cm mass was noted on the tip of the posterior mitral valve leaflet with findings consistent with NBTE. He had moderate to severe, central mitral regurgitation (MR) and an intact interatrial septum with no evidence of a shunt by color Doppler or with injection of agitated saline. The left atrial appendage appeared normal. The mitral valve and bioprosthetic aortic valve were noted to have been functioning well at an outside hospital transthoracic echocardiogram (TTE) report a few years back. Other significant lab results are presented in. IgG B2 Glycoprotein 1 Antibody and IgG Cardiolipin Antibody were elevated consistent with his history of antiphospholipid syndrome. Mitral valve endocarditis was managed conservatively with anticoagulation. He was discharged to a subacute rehab on subcutaneous enoxaparin 100 mg two times daily. Patient had a subsequent hospitalization 3 months later with a fever of 100.9°F and a low hemoglobin of 6.7 g/dl (reference range: 14-17.5 g/dL). He was noted to have hemolytic anemia with reticulocytes of 10.9% (reference range: 0.5-2.0%), mild lactose dehydrogenase elevation of 287 IU/L (reference range: 140-271 IU/ L), and elevated indirect bilirubin of 1 mg/dL (calculated by subtracting direct from total bilirubin). His direct Coombs test was positive for warm autoantibody IgG and C3 complement. His fecal occult blood test was negative. He received blood transfusion as necessary with goal hemoglobin > 7.0 g/dl and was started on intravenous methylprednisone that was tapered to hydrocortisone due to his adrenal insufficiency. Two out of 2 of his blood cultures from admission came back positive for Enterococcus faecalis which cleared on the sixth day of admission with antibiotics. Patient had a chronic indwelling Foley's catheter. Source of bacteremia was urine which grew >100,000 CFU/mL E. faecalis and >100,000 CFU/mL Escherichia coli. The patient was placed on intravenous vancomycin and ceftriaxone. Vancomycin was later changed to daptomycin due to persistent bacteremia. TTE showed focal thickening and calcification of the mitral valve leaflets and subvalvular apparatus, a small density on the atrial side of the mitral valve and moderate to severe mitral regurgitation. TEE further showed 0.6 × 0.5 cm and 0.4 × 0.3 cm masses attached to the tip of the anterior and posterior MV leaflets, respectively, with moderate to severe, central MR. No obvious vegetation was noted in the aortic valve. Given persistent bacteremia and severe MR, patient underwent redo sternotomy and mitral valve replacement with 27 mm Edwards bioprosthesis. Endocarditic breakdown of mitral valve was observed but no real vegetation was noted during the procedure. Histologic examination of the mitral valve was consistent with vegetation, but bacterial, fungal, and anaerobic cultures were negative for definitive organisms. Post-operative stay was uneventful, and patient was discharged on IV daptomycin to complete a 6-week course. The patient was hospitalized a month later with right temporal occipital intraparenchymal hemorrhage measuring 4.4 × 3.8 × 3.2 cm with adjacent edema but no midline shift. This was thought to be due to hemorrhagic conversion of prior stroke. Hemorrhagic stroke was managed conservatively by holding enoxaparin for 4 days and starting levetiracetam for seizure prophylaxis. He was again hospitalized a month and a half later with 3.8 × 3.4 × 2.7 cm large right cerebellar hematoma with mass effect on the fourth ventricle without hydrocephalus. Patient passed away later the same day thought to be from brain herniation. # Discussion ## Pathogenesis Epithelial injury is believed to be the inciting factor. This is followed by fibrin and platelet deposition of the affected valve in the setting of a hypercoagulable state. Antiphospholipid antibodies and complement have been noted on involved valves which suggests that APS is a risk factor for NBTE. It is believed that these autoantibodies are directed against the phospholipids in the endothelium and not only damage the endothelium but also mediate activation and deposition of platelet and fibrin. These lesions are more friable than the vegetations seen in infective endocarditis and are more prone to systemic embolization. ## Clinical presentation NBTE is typically clinically silent and patients commonly present with symptoms of embolization or valvular dysfunction. Patients may embolize to the brain, extremities, coronaries, spleen, and kidneys which manifests as focal neurological deficits, memory loss, painful extremities, and acute abdomen. Cerebral embolization is the most common clinical manifestation of APS in patients with NBTE. Vavlular dysfunction may present as heart murmurs, arrhythmias, and heart failure. Patients with primary APS may also have history of multiple miscarriages and arterial and venous thromboses. ## Diagnosis Anticardiolipin IgG or IgM, anti-B 2 glycoprotein IgG or IgM and lupus anticoagulant associated with history of thrombosis or pregnancy morbidity are required for a diagnosis of definite APS. Patients may also have hemolytic anemia and thrombocytopenia. Schistocytes may also be present. Positive antiphospholipid antibodies, negative blood cultures, and absence of systemic symptoms help differentiate NBTE from infective endocarditis. Mitral valve followed by aortic valve are commonly affected valves. It can affect both normal valves and valves affected by rheumatic fever or endocarditis, chordae tendineae or the endocardium. Valvular lesions are characterized by localized to generalized thickening with or without vegetations. Vegetations are often rounded, sessile, and heterogenous and are characteristically seen along the coaptation edge of the leaflets. There may be associated valvular regurgitation or stenosis. All these findings can be detected on TTE or TEE. Definitive diagnosis can be done by biopsy of the affected valve or postmortem. Histologic examination shows fibrinoplatelet deposition with mononuclear cell infiltration. ## Management No specific therapy exists for NBTE. Treatment should be directed at preventing systemic embolization and management of underlying malignancy or SLE. Long-term anticoagulation is recommended by the American College of Chest Physician guidelines. Unfractionated heparin is currently recommended to reduce rates of ischemic stroke in cancer patients. Limited evidence exists on the use of direct oral anticoagulants in these patients. Role of steroids is unclear but has shown to decrease the prevalence of Libman-Sacks endocarditis. Patients with severe symptomatic valvular dysfunction, recurrent systemic embolism, and large vegetations greater than 1 cm may undergo valve repair or replacement after assessing their individual risks and benefits. The prognosis of patients with NBTE is poor. # Conclusion Our patient is a unique case of recurrent NBTE with multiple valve replacements that were complicated by recurrent ischemic strokes despite being on anticoagulation. We hope this case will help better understand treatment for similar cases of recurrent NBTE and strokes.

Photosynthetic and Canopy Characteristics of Different Varieties at the Early Elongation Stage and Their Relationships with the Cane Yield in Sugarcane During sugarcane growth, the Early Elongation stage is critical to cane yield formation. In this study, parameters of 17 sugarcane varieties were determined at the Early Elongation stage using CI-301 photosynthesis measuring system and CI-100 digital plant canopy imager. The data analysis showed highly significant differences in leaf area index (LAI), mean foliage inclination angle (MFIA), transmission coefficient for diffused light penetration (TD), transmission coefficient for solar beam radiation penetration (TR), leaf distribution (LD), net photosynthetic rate (PN), transpiration rate ( ), and stomatal conductance (GS) among sugarcane varieties. Based on the photosynthetic or canopy parameters, the 17 sugarcane varieties were classified into four categories. Through the factor analysis, nine parameters were represented by three principal factors, of which the cumulative rate of variance contributions reached 85.77%. A regression for sugarcane yield, with relative error of yield fitting less than 0.05, was successfully established: sugarcane yield = −27.19 − 1.69 × PN + 0.17 × + 90.43 × LAI − 408.81 × LD + 0.0015 × NSH + 101.38 × ( 2 = 0.928 * * ). This study helps provide a theoretical basis and technical guidance for the screening of new sugarcane varieties with high net photosynthetic rate and ideal canopy structure. # Introduction Photosynthesis is the basis of yield formation. It is generally recognized that about 90%-95% of crop yield is formed from the assimilated carbons. Therefore, how to improve crops' photosynthetic capacity is one of the most important targets in the studies of genetics and breeding [bib_ref] Studies on cultivated physiological indexes for high-yielding cotton in Xinjiang, Wang [/bib_ref] [bib_ref] The compensatory mechanism in exploring crop production potential, Zhao [/bib_ref] [bib_ref] Progress in ideotype breeding to increase rice yield potential, Peng [/bib_ref] [bib_ref] A comparison of three isolines of cotton diff ering in fiber color..., Hua [/bib_ref]. The canopy structure of a crop should be optimized so that an increased proportion of light can reach the leaves at the base of the plants [bib_ref] Spatial distribution of leaf and boll in relation to canopy photosynthesis of..., Feng [/bib_ref] [bib_ref] Can improvement in photosynthesis increase crop yields, Long [/bib_ref]. Studies on photosynthetic characteristics of crops such as Zea mays [bib_ref] Canopy structure and photosynthetic characteristics of high yield and high nitrogen efficiency..., Jin [/bib_ref] [bib_ref] Cluster analysis for photosynthetic characters of inbred lines of maize in china, Zhao [/bib_ref] [bib_ref] Studies on photosynthetic properties of different maize inbred lines, Guo [/bib_ref] , Oryza sativa [bib_ref] Varietal difference in photosynthetic characteristics of rice under photooxidation and shading, Li [/bib_ref] [bib_ref] Erect leaves and photosynthesis in rice, Sinclair [/bib_ref] [bib_ref] Analysis on combining ability and heritability of photosynthetic characters in indica hybrid..., Zhai [/bib_ref] , and Glycine max [bib_ref] Development of soybean cultivars (germplasm) with high photosynthetic efficiency (HPE) and rediscussion..., Du [/bib_ref] [bib_ref] The high photosynthetic efficiency characteristics of high-yield varieties in soybean, Li [/bib_ref] have been extensively performed. Also, inheritance of photosynthetic characteristics [bib_ref] Analyses for inheritance and combining ability of photochemical activities measured by chlorophyll..., Zhang [/bib_ref] [bib_ref] Genetic variation of net photosynthetic rate in sugarcane hybrid progenies, Gao [/bib_ref] [bib_ref] Study on the genetic analysis of parents with high photosynthetic efficiency in..., Lü [/bib_ref] , diurnal variation of net photosynthetic rate [bib_ref] Diurnal variation of photosynthetic rate in sugarcane and its responses to light..., Zhang [/bib_ref] [bib_ref] Effect of ethephon on respiratory exchange in different sugarcane varieties, Xing [/bib_ref] , and seasonal variation [bib_ref] Seasonal change of the net photosynthesis rate, chlorophyll content and specific weight..., Lü [/bib_ref] [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref] have been reported in the separated sugarcane seedling populations. Indices such as shape, size, quantity, and spatial distribution of leaves are directly related to the light environment and light utilization rate of the population. They are the important factors affecting the light distribution in crop population and photosynthesis [bib_ref] Genetic analysis of main economic and photosynthetic traits in energy sugarcane, Luo [/bib_ref]. Optimizing a reasonable population structure, improving the light distribution in the population, and increasing the rate of light utilization are all effective ways in obtaining high crop yields. Leaf area index (LAI) determines canopy light interception, which affects the photosynthetic rate of a crop population. When LAI is at the optimal level, canopy light interception reaches the highest level for improved photosynthetic capacity and potential yield increase [bib_ref] Regulating effects of density and top-dressing time of nitrogen on characterstics of..., Zhao [/bib_ref]. The relations between sugarcane leaf morphology and cane yield and between sucrose content and transpiration rate of leaves have been studied [bib_ref] Mathematical analysis on leaf morphology of sugarcane varieties, Gao [/bib_ref] [bib_ref] Studies on the relationship of morphological, anatomical characters in seedling stage and..., Tan [/bib_ref] [bib_ref] Morphophysiological responses of sugarcane genotypes to water stress, Zhang [/bib_ref]. However, studies on the canopy characteristics of sugarcane were rarely reported [bib_ref] Factor and clustering analysis of economic characters in sugarcane(Saccharum spp, Gao [/bib_ref] [bib_ref] Interspecific variation in physiology of conifer and analysis by fuzzy cluster, Zhang [/bib_ref] , [bib_ref] The compensatory mechanism in exploring crop production potential, Zhao [/bib_ref] The Scientific World Journal although there are many yield-related traits and indices controlling photosynthesis and canopy structure of sugarcane. The interactions between these indices are complicated due to the influences of genetic characteristics, environmental factors, and sample selections. The factor analysis method allows the categorization of a large number of correlated agronomic traits into several groups based on principal factors. It has been widely used in studies of crop germplasm resources. In sugarcane, a correlation analysis of seasonal variation of the canopy structure parameters to yield-related traits was conducted [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref] ; however, there has been no report on the application of factor analysis to the classification of photosynthetic and canopy parameters and their effects on crop yield in sugarcane [bib_ref] Data Processing System (DPS) software with experimental design, statistical analysis and data..., Tang [/bib_ref] [bib_ref] Phenetic classification for photosynthetic characters of different sugarcane varieties, Luo [/bib_ref]. During sugarcane growth, Early Elongation stage is a critical period for cane yield formation. During this period, an appropriate number of stalks per unit area are controlled through timely cultivation to achieve high yield. In this study, we aimed to investigate the effects of photosynthetic and canopy characteristics of 17 sugarcane varieties at the Early Elongation stage on cane yield. The results of this study would provide the scientific basis for breeding of sugarcane varieties with high photosynthetic efficiency and for improving cultivation technique. Hierarchical cluster analysis was performed by the numerical classification method on the photosynthetic and canopy parameters of the involved sugarcane varieties. The relationship and the stepwise regression analysis between canopy and photosynthetic characteristics and cane yield were also conducted, in order to screen new sugarcane varieties with high net photosynthetic rate (PN) and ideal canopy structure. [fig_ref] Table 1: Cane yield traits of 17 sugarcane varieties [/fig_ref] -835, and YT96-86. # Materials and methods ## Sugarcane varieties. ## Field experiment design and cane yield traits. Field experiment was conducted on the Experimental Farm of the Key Laboratory of Sugarcane Biology and Genetic Breeding, Ministry of Agriculture/Fujian Agriculture and Forestry University located at Jianxin, Cangshan District, Fuzhou, Fujian (longitude: 119.23E, latitude: 26.08N). A randomized block design with three replications was adopted. Plot area was 33 m 2 , with three rows at 1.1 m row spacing. Planting density was 45,000 two-bud setts ha −1 . The preceding crop was sugarcane, and the soil type was sandy loam soil. The nutrients of the arable layer before sowing included organic matter of 10.5 g/kg, total nitrogen of 0.91 g/kg, alkaline hydrolysis nitrogen of 90.01 mg/kg, available phosphorus of 110.4 mg/kg, and rapidly available potassium of 369.5 mg/kg. The rows were covered by plastic films after planting. Planting was performed in a scheme of double buds and double rows. Herbicides were applied before planting and plastic film mulching. Weed control was repeatedly performed as needed. The basal fertilizer Calcium Super Phosphate at 750 kg ha −1 was applied during planting. During growing season, 975 kg ha −1 and 600 kg ha −1 KCl were applied for two times as topdressing. Field management level was only slightly more intensive than that of the local standard operations on cultivation, fertilization, irrigation, and pest control. Each application was completed covering all the experimental plots on the same day. In Fuzhou, sugarcane elongation period lasts from late June to early November, while Early Elongation stage commences from late June and ends in early July. Measurements of photosynthetic characteristics and canopy parameters were conducted in early July when sugarcane seedlings reached an average height of 80.9 cm. Data on yield-related traits, including plant height ( ), stalk diameter ( ), single stalk weight (SSW), and the number of effective stalks per hectare (NSH), were collected before harvesting. All the stalks in the middle row of each plot were cut and weighed. The sampled sugarcane areas were measured. The number of millable stalks within the sampling area was also counted. Single stalk weight and cane yield were calculated by the following formulae: Single stalk weight = (plant height × stalk diameter 2 × 0.785)/1000; Cane yield = single stalk weight × the number of effective stalks per hectare. ## Determination of photosynthetic parameters. Photosynthetic parameter data were collected between 8:30-11:30 AM on a sunny morning in early July, using a CI-301 photosynthesis system (CID Co., Ltd, Vancouver, WA, USA) under natural light conditions. The data included net photosynthetic rate (PN, molm −2 s −1 ), transpiration rate ( , mmolm −2 s −1 ), and stomatal conductivity (GS, mmolm −2 s −1 ). The PN equals the rate of photosynthetic CO 2 fixation minus the rate of CO 2 loss during respiration. The is the amount of evaporation per unit time from a leaf surface. The GS is the rate of CO 2 entering stomata [bib_ref] Study on the genetic analysis of parents with high photosynthetic efficiency in..., Lü [/bib_ref] [bib_ref] Genetic analysis of main economic and photosynthetic traits in energy sugarcane, Luo [/bib_ref]. The measurements were taken three times on each variety following the protocol described earlier with minor modifications [bib_ref] Study on the genetic analysis of parents with high photosynthetic efficiency in..., Lü [/bib_ref] [bib_ref] Genetic analysis of main economic and photosynthetic traits in energy sugarcane, Luo [/bib_ref] [bib_ref] Phenetic classification for photosynthetic characters of different sugarcane varieties, Luo [/bib_ref]. The first youngest fully expanded (+1) leaves at the top canopy were measured reciprocally at the middle to upper section excluding the midrib. The direction of leaf chamber was adjusted towards sunlight to ensure that measurements were done under a uniform light intensity. A total of 18 plants were measured for each variety under the following natural conditions: light intensity (1,781.17 ± 103.21 molm −2 s −1 ), ambient temperature (T ) (28.07 ± 2.44 ∘ C), leaf temperature (30.14 ± 2.26 ∘ C), ambient relative humidity (RH) (47.71 ± 4.88%), and ambient CO 2 concentration (C ) (331.02 ± 25.35 L⋅L −1 ). Several parameters were recorded simultaneously, including effective photosynthetic radiation (PAR), relative humidity (RH), ambient temperature (T ), ambient CO 2 concentration (C ), stomatal conductivity (GS), evaporation rate ( ), and between cell CO 2 concentrations (C ). Because all determinations were done under the natural sunlight, only those measurements were kept when natural light intensity is at 1,600 mol/M 2 ⋅Sec, while measurements taken under 1,600 mol/M 2 ⋅Sec light intensity were discarded. CO 2 concentrations were collected from the upper level of canopy after passing through the dual-bottle apparatus and were slightly lower than those collected above the ground level. This is due to the fact that sugarcane is a highly efficient C 4 crop. Only mean values and standard deviations were shown in the text. ## Determination of canopy parameters. A fisheye lens of the CI-100 digital plant canopy imager (CID Co., Ltd, Vancouver, WA, USA) was attached to an observation rod and placed in the center of the rows in a cloudless twilight afternoon with a little sunshine in early July. The rod was adjusted to the horizontal direction for taking photos when there was no shadow or any other external influences. Fifteen testing points were selected for each variety and five images were taken from each plot. LD was represented by distribution frequency of the leaf within each azimuth. The canopy indicators such as leaf area index (LAI), mean foliage inclination angle (MFIA), transmission coefficient for diffuse light penetration (TD), transmission coefficient for solar beam radiation penetration (TR), and leaf distribution (LD) were calculated by Plant Canopy Analysis software provided by CID Company (CID Co., Ltd, Vancouver, WA, USA) [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref]. ## Statistical Methods. Variance analysis, cluster analysis, factor analysis, and regression analysis were performed using DPS software (Zhejiang University, Hangzhou, China) [bib_ref] Data Processing System (DPS) software with experimental design, statistical analysis and data..., Tang [/bib_ref]. After standardization of original data, the distance coefficient was determined as the chi-square distance and cluster analysis by Ward's method [bib_ref] Data Processing System (DPS) software with experimental design, statistical analysis and data..., Tang [/bib_ref]. Varimax rotation was used to change the coordinates used in principal component analysis and factor analysis [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref] [bib_ref] Data Processing System (DPS) software with experimental design, statistical analysis and data..., Tang [/bib_ref]. A stepwise regression analysis was also conducted for the photosynthetic and canopy parameters and their correlation with cane yield [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref] [bib_ref] Data Processing System (DPS) software with experimental design, statistical analysis and data..., Tang [/bib_ref]. # Results ## Estimated cane yield trait data. Estimated cane yield trait data are shown in [fig_ref] Table 1: Cane yield traits of 17 sugarcane varieties [/fig_ref]. ## Differences in photosynthetic ## Differences in the canopy parameters among 17 sugarcane Varieties. There were highly significant differences in LAI, MFIA, TD, TR, and LD among the 17 sugarcane varieties at the Early Elongation stage ( Hierarchical cluster analysis was performed by merging all canopy parameters' data, that is, LAI, MFIA, TD, TR, and LD, and the output was shown in . The 17 varieties again were grouped into four clusters (Table 5, : A clustering dendrogram based on canopy parameters of 17 sugarcane varieties. [fig_ref] Table 6: Loading matrix of characters initial factor in sugarcane varieties [/fig_ref]. The cumulative contribution rates of the characteristic values of the first three principal factors reached 85.77%. [fig_ref] Table 6: Loading matrix of characters initial factor in sugarcane varieties [/fig_ref] also indicates that the essential information of the first three principal factors can reflect most of the information of these photosynthetic gas exchange parameters, canopy parameters, and yield-related traits. The nine traits had much in common with a high degree of freedom. It also revealed that these three principal factors represented the nine traits well. The factor loading matrices after varimax rotation in three principal factors were shown in [fig_ref] Table 7: Loading matrix of varimax orthogonal rotation factor in sugarcane varieties [/fig_ref]. By comparison between the factor loading matrices of postvarimax rotation [bib_ref] Can improvement in photosynthesis increase crop yields, Long [/bib_ref] The Scientific World Journal and the ones of prevarimax rotation, the load values of important variables in the principal factors were increased significantly. The biological significance of the principal factors after the varimax rotation also became more significant. As can be seen from [fig_ref] Table 7: Loading matrix of varimax orthogonal rotation factor in sugarcane varieties [/fig_ref] , the large load values for canopy parameters, LAI, MFIA, TD, TR, and LD, indicated that these values played a major role in the first principal factor. The canopy parameters also had a close relationship with cane yield. On the other hand, the load values of the photosynthetic parameters PN, , and GS were relatively large in the second principal factor, while the load values of SSW and NSH were relatively large in the third principal factor. Collectively, these factors were entitled as the yield component factors. The correlation analyses data listed in [fig_ref] Table 8: Correlation matrix among the traits in sugarcane varieties [/fig_ref] indicated the following: (1) PN had a positive correlation with and GS; (2) LAI had a negative correlation with MFIA, TD, and TR, but a significantly positive correlation with NSH; (3) cane yield had a significantly negative correlation with MFIA, TD, and TR, but a significantly positive correlation with LD; and (4) SSW had significantly negative correlation with NSH, but a significantly positive correlation with . Based on regression analysis, cane yield was predictable based on photosynthetic and canopy parameters. The regression equation is as follows: Cane yield = − 27.19 − 1.69 × PN + 0.17 × + 90.43 × LAI − 408.81 × LD + 0.0015 × NSH + 101.38 × ( 2 = 0.928 * * ). The relative error of cane yield fitting was less than 5% for all the 17 sugarcane varieties. Parameters NSH and play a decisive role in cane yield formation with highly significant correlation coefficients of 0.902 and 0.94. Parameters LAI and LD also play a major role with significant partial correlation coefficients of 0.682 and 0.624. However, parameters PN and only influence the formation of cane yield slightly with nonsignificant partial correlation coefficients of −0.331 and 0.298. Based on these observations, we conclude that the Early Elongation (growth) stage is critical to the life cycle of sugarcane. It is during this elongation stage that NSH and start to form a canopy structure, which in turn determines the yield level of photosynthetic products. As was discussed in our previous report [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref] , cane yield formation could be influenced by many factors, which need to be adjusted according to canopy structure to ensure final cane yield formation. # Discussion and conclusions Regarding the genotypic differences in photosynthetic capacities among sugarcane varieties, the PN and GS could be affected greatly by the specific combining ability among sugarcane varieties [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref]. However, the correlation coefficient The Scientific World Journal 7 between F 1 hybrids and their parents reached a significant level in terms of general combining ability. Therefore, parental varieties with high photosynthetic efficiency should be selected in sugarcane breeding [bib_ref] Genetic analysis of main economic and photosynthetic traits in energy sugarcane, Luo [/bib_ref]. Sugarcane breeding practice demonstrates that PN and GS (water use efficiency) can be combined. Although the photosynthetic parameters PN, , and GS had high degrees of separations in F 1 seedling populations [bib_ref] Study on the genetic analysis of parents with high photosynthetic efficiency in..., Lü [/bib_ref] , the high photosynthetic efficiency, once generated upon cross-recombination, can be immobilized by asexual reproduction, which is a unique feature of sugarcane production. This is why all the advanced varieties possess good PN, , and GS parameters [bib_ref] Phenetic classification for photosynthetic characters of different sugarcane varieties, Luo [/bib_ref]. In the present study, there were significant differences in the photosynthetic parameters among the 17 sugarcane varieties at the Early Elongation stage, including PN, , and GS, which are in accordance with the previous reports [bib_ref] Study on the genetic analysis of parents with high photosynthetic efficiency in..., Lü [/bib_ref] [bib_ref] Genetic analysis of main economic and photosynthetic traits in energy sugarcane, Luo [/bib_ref] [bib_ref] Phenetic classification for photosynthetic characters of different sugarcane varieties, Luo [/bib_ref]. The sugarcane varieties were classified according to the three photosynthetic traits, respectively, and the comprehensive traits and natural types were well reflected. The clustering patterns were relatively stable, although the masking of some traits by others and some ambiguous intercategory differences did exist. Therefore, single and integrated traits should be combined in photosynthetic efficiency breeding to obtain a better product [bib_ref] Cluster analysis for photosynthetic characters of inbred lines of maize in china, Zhao [/bib_ref]. Leaf area determines the canopy light interception. The distribution of leaf area within canopy is an important index of crop canopy structure [bib_ref] Relationship between canopy characters and leaf morphology at different positions of sugarcane, Luo [/bib_ref]. Previous studies showed that there were significant differences in canopy parameters LAI, MFIA, and TD at the seedling stage among different genotypes. Varieties with large LAI and small TD at the seedling 8 The Scientific World Journal stage had a higher number of leaves per unit space. A positive relationship exists between even leaf spatial distribution and light interception efficiency of the population and hence higher cane yield [bib_ref] The Relationship of canopy structure characters in the seedling and yield characters..., Luo [/bib_ref]. Leaf canopy structure directly affects the canopy light interception and light distribution at different layers. Researches on morphology and anatomy revealed that sugarcane populations with transparent canopy of short, narrow, thick, and straight leaves had a larger yield potential. In addition, LAI has a very close relationship with dry matter accumulation. Therefore, a proper increase of the population LAI can increase dry matter accumulation [bib_ref] Relationship between canopy characters and leaf morphology at different positions of sugarcane, Luo [/bib_ref] [bib_ref] The Relationship of canopy structure characters in the seedling and yield characters..., Luo [/bib_ref] [bib_ref] Study on canopy structure and physiological characteristics of super-high yield spring maize, Zhang [/bib_ref] [bib_ref] Effect of density on canopy structure and photosynthetic characteristics in soybean population, Zhang [/bib_ref] [bib_ref] Canopy structure and light distribution in winter wheat, Zhang [/bib_ref]. In the present study, MFIA affects the receiving of solar radiation and light distribution in canopy. The ideal leaf population structure has the most effective leaf area with the characteristics of continuously changing the inclination angle distribution. Canopy structure and light distribution of sugarcane at the Early Elongation stage play a crucial role in the accumulation and distribution of photosynthetic products, in the growth and development of population, and in the formation of final products [bib_ref] Morphophysiological responses of sugarcane genotypes to water stress, Zhang [/bib_ref] [bib_ref] Factor and clustering analysis of economic characters in sugarcane(Saccharum spp, Gao [/bib_ref] [bib_ref] Interspecific variation in physiology of conifer and analysis by fuzzy cluster, Zhang [/bib_ref] [bib_ref] Data Processing System (DPS) software with experimental design, statistical analysis and data..., Tang [/bib_ref]. This has been clearly explained by the correlations between the main canopy parameters and cane yield in this study. Zhang et al. [bib_ref] Effect of density on canopy structure and photosynthetic characteristics in soybean population, Zhang [/bib_ref] showed that canopy MFIA of winter wheat decreased with the increase of nitrogen content of soil. The light distributions of two crop populations with the same LAI are different, if the spatial distributions of MFIA and leaves are different. They can lead to different photosynthetic rates of canopy and different amounts of dry matters were produced by populations per unit time [bib_ref] Effect of density on canopy structure and photosynthetic characteristics in soybean population, Zhang [/bib_ref]. However, further optimization is still required when the yield-related traits are indirectly selected based on the main canopy parameters since the yield of sugarcane has a negative correlation with quality-related traits in this study. What should also be stressed is that, for those varieties with large LAI at the Early Elongation stage, the LAI should be adjusted to be within a certain range at the middle to late stages. Mutual shading among leaves should be avoided, which weakens light intensity, reduces light interception, and decreases photosynthetic efficiency. Therefore, rapid and accurate diagnosis of canopy structure of sugarcane and a timely, reasonable regulation of population size play important roles in the cultivation of high-quality and highyield sugarcane varieties [bib_ref] Relationship between canopy characters and leaf morphology at different positions of sugarcane, Luo [/bib_ref] [bib_ref] The Relationship of canopy structure characters in the seedling and yield characters..., Luo [/bib_ref]. During the Early Elongation stage, the photosynthetic and canopy parameters play a crucial role in cane yield formation [bib_ref] Seasonal variation of the canopy structure parameters and its correlation with yieldrelated..., Luo [/bib_ref]. In this study, highly significant differences in LAI, MFIA, TD, TR, LD, PN, , and GS were observed among 17 sugarcane varieties at the Early Elongation stage. However, during the factor analysis process, nine parameters were expressed by three principal factors, of which the cumulative variance contribution rate reached 85.77%. Both correlation analysis and factor analysis showed that, at the Early Elongation stage, there were close relationships between photosynthetic and canopy parameters and cane yield. Based on regression analysis for sugarcane yield, with relative error of yield fitting less than 5%, a cane yield prediction formula was established: Cane yield = − 27.19 − 1.69 × PN + 0.17 × + 90.43 × LAI − 408.81 × LD + 0.0015 × NSH + 101.38 × ( 2 = 0.928 * * ), in which photosynthetic parameters, such as PN and , and canopy parameters, such as LAI and LD, are used as the indirect indices for selecting better yield-related traits at the Early Elongation stage. From all the above, this study should provide the theoretical foundation and technical guidance for identifying new sugarcane varieties with high photosynthetic rates and ideal canopy structures. [table] Table 1: Cane yield traits of 17 sugarcane varieties. [/table] [table] Table 2: Photosynthesis rate (PN), transpiration rate ( ), and stomatal conductivity (GS) of 17 sugarcane varieties. [/table] [table] Table 3: Differences among different photosynthetic gas exchange parameters among 17 sugarcane varieties. which shared relatively high PN, , and GS values. Cluster II included five varieties, that is, FN94-0744, GT94-116, GT95-118, GT97-18, and YT92-1287. The PN values of these varieties were at middle levels, while all had high and GS values. Cluster III included four varieties (MT70-611, ROC10, YT96-107, and YT96-835) with middle PN, low , and low GS values. Cluster IV only had one variety, GT96-44, with low values of PN, , and GS. [/table] [table] Table 4: According to LAI, 17 sugarcane varieties were classified into three categories. Category I included six large LAI varieties FN94-0403, GT94-116, FN96-0907, FN98-10100, GT96-44, and MT70-611, with a mean value 1.35. Category II included 4 middle LAI varieties FN94-0744, GT96-211, GT97-18, and YT92-1287, with a mean value 1.155. Category III included seven low LAI varieties FN95-1726, GT95-118, ROC10, YT96-107, YT96-794, YT96-835, and YT96-86, with a mean value 1.033. [/table] [table] Table 5: Difference in different canopy parameters among 17 sugarcane varieties. [/table] [table] Table 6: Loading matrix of characters initial factor in sugarcane varieties. [/table] [table] Table 7: Loading matrix of varimax orthogonal rotation factor in sugarcane varieties. [/table] [table] Table 8: Correlation matrix among the traits in sugarcane varieties. TD: transmission coefficient for diffuse penetration; TR: transmission coefficient for solar beam radiation penetration; LD: leaf distribution; CY: cane yield; NSH: number of stalks per hectare; D: diameter; SSW: fresh single stalk weight. (2) − Negative load value; the larger absolute value means the larger load value. (3) * Significant differences at the level of 0.05; * * significant differences at the level of 0.01. [/table]

Predictive Power of a Body Shape Index for Development of Diabetes, Hypertension, and Dyslipidemia in Japanese Adults: A Retrospective Cohort Study Background/ObjectivesRecently, a body shape index (ABSI) was reported to predict all-cause mortality independently of body mass index (BMI) in Americans. This study aimed to evaluate whether ABSI is applicable to Japanese adults as a predictor for development of diabetes, hypertension, and dyslipidemia.Subjects/MethodsWe evaluated the predictive power of ABSI in a retrospective cohort study using annual health examination data from Chiba City Hall in Japan, for the period 2008 to 2012. Subjects included 37,581 without diabetes, 23,090 without hypertension, and 20,776 without dyslipidemia at baseline who were monitored for disease incidence for 4 years. We examined the associations of standardized ABSI, BMI, and waist circumference (WC) at baseline with disease incidence by logistic regression analyses. Furthermore, we conducted a casematched study using the propensity score matching method.ResultsElevated BMI, WC, and ABSI increased the risks of diabetes and dyslipidemia [BMI-diabetes: odds ratio (OR) = 1.26, 95% confidence interval (95%CI) = 1.20−1.32; BMI-dyslipidemia: OR = 1.15, 95%CI = 1.12−1.19; WC-diabetes: OR = 1.24, 95%CI = 1.18−1.31; WC-dyslipidemia: OR = 1.15, 95%CI = 1.11−1.19; ABSI-diabetes: OR = 1.06, 95%CI = 1.01 −1.11; ABSI-dyslipidemia: OR = 1.04, 95%CI = 1.01−1.07]. Elevated BMI and WC, but not higher ABSI, also increased the risk of hypertension [BMI: OR = 1.32, 95%CI = 1.27−1.37; WC: OR = 1.22, 95%CI = 1.18−1.26; ABSI: OR = 1.00, 95%CI = 0.97−1.02]. Areas under the curve (AUCs) in regression models with ABSI were significantly smaller than in models with BMI or WC for all three diseases. In case-matched subgroups, the power of ABSI was PLOS ONE | # Introduction Obesity is a well-known risk factor for mortality from all causes [bib_ref] Bodymass index and mortality among 1.46 million white adults, Berrington De Gonzalez [/bib_ref] [bib_ref] Association of all-cause mortality with overweight and obesity using standard body mass..., Flegal [/bib_ref] and cardiovascular disease (CVD) [bib_ref] Bodymass index and mortality among 1.46 million white adults, Berrington De Gonzalez [/bib_ref] [bib_ref] Association of obesity with cardiovascular disease mortality in the PLCO trial, Jiang [/bib_ref] , and for incidences of non-communicable diseases such as diabetes [bib_ref] Effect of obesity on incidence of type 2 diabetes declines with age..., Fujita [/bib_ref] [bib_ref] Obesity, weight change and risks for hypertension, diabetes and hypercholesterolemia in Japanese..., Ishikawa-Takata [/bib_ref] , hypertension [bib_ref] Obesity, weight change and risks for hypertension, diabetes and hypercholesterolemia in Japanese..., Ishikawa-Takata [/bib_ref] [bib_ref] Incidence of hypertension in a prospective cohort study of adults from Porto,..., Pereira [/bib_ref] , and dyslipidemia [bib_ref] Obesity, weight change and risks for hypertension, diabetes and hypercholesterolemia in Japanese..., Ishikawa-Takata [/bib_ref]. Although body mass index (BMI, weight in kilograms divided by the square of height in meters) is widely used as a measure of obesity, it has potential weaknesses. The BMI does not distinguish weight due to fat accumulation from muscle weight [bib_ref] Relationship between adiposity and body size reveals limitations of BMI, Nevill [/bib_ref] [bib_ref] Body mass index classification misses subjects with increased cardiometabolic risk factors related..., Gómez-Ambrosi [/bib_ref] and does not distinguish peripheral fat from abdominal fat [bib_ref] Trunk fat is associated with increased serum levels of alanine aminotransferase in..., Ruhl [/bib_ref] , the latter being more strongly associated with CVD risk. To partly overcome these weaknesses, waist circumference (WC) is used as an indicator of abdominal fat accumulation, and many studies have shown that WC can predict mortality from all causes and CVD more accurately than BMI [bib_ref] Body mass index, waist circumference and waist-hip ratio: which is the better..., Czernichow [/bib_ref] [bib_ref] Preferred clinical measures of central obesity for predicting mortality, Welborn [/bib_ref]. However, WC reflects not only abdominal fat accumulation but also overall body size (height and weight). In fact, WC is strongly correlated with both weight and BMI [bib_ref] Waist versus weight: which matters more for mortality?, Moore [/bib_ref] [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref]. Krakauer et al. described a new anthropometric measure, a body shape index (ABSI), which quantifies abdominal adiposity relative to BMI and height, and reported that ABSI predicted all-cause mortality independently of BMI in a large cohort of American adults during an average 4.8-year follow-up [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref]. They also indicated that the predictive power of ABSI for all-cause mortality differed among ethnicities, with less predictive power in Latinos than Caucasian and African Americans, and acknowledged that additional cohort studies with other ethnic groups are necessary to clarify the limits of ABSI predictive efficacy [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref]. To date, six cohort studies evaluating the ABSI for prediction of mortality or morbidity have been published [bib_ref] Comparison of various surrogate obesity indicators as predictors of cardiovascular mortality in..., Song [/bib_ref] [bib_ref] Dynamic association of mortality hazard with body shape, Krakauer [/bib_ref] [bib_ref] Body shape index and mortality in hemodialysis patients, Afsar [/bib_ref] [bib_ref] Association between different obesity measures and the risk of stroke in the..., Abete [/bib_ref] [bib_ref] Could the new body shape index predict the new onset of diabetes..., He [/bib_ref] [bib_ref] A Body Shape Index" in middle-age and older Indonesian population: scaling exponents..., Cheung [/bib_ref]. Of these, three described the predictive power of ABSI for mortality from CVD [bib_ref] Comparison of various surrogate obesity indicators as predictors of cardiovascular mortality in..., Song [/bib_ref] and all causes [bib_ref] Dynamic association of mortality hazard with body shape, Krakauer [/bib_ref] [bib_ref] Body shape index and mortality in hemodialysis patients, Afsar [/bib_ref]. The largest of these studies, including 46,651 Europeans, concluded that the predictive powers of several abdominal obesity indicators, such as WC, waist-to-hip ratio, and waist-tostature ratio, were stronger predictors of CVD mortality than were BMI and ABSI [bib_ref] Comparison of various surrogate obesity indicators as predictors of cardiovascular mortality in..., Song [/bib_ref] , while a British study of 7,011 adults reported that ABSI was a robust predictor of all-cause mortality [bib_ref] Dynamic association of mortality hazard with body shape, Krakauer [/bib_ref]. The smallest study, with 142 hemodialysis patients, found no relation between ABSI and mortality [bib_ref] Body shape index and mortality in hemodialysis patients, Afsar [/bib_ref]. In the remaining three studies, the principle outcome measure was morbidity. One showed that ABSI was significantly associated with total stroke incidence in men, while BMI was not [bib_ref] Association between different obesity measures and the risk of stroke in the..., Abete [/bib_ref]. The others found that ABSI was associated with development of diabetes [bib_ref] Could the new body shape index predict the new onset of diabetes..., He [/bib_ref] or hypertension [bib_ref] A Body Shape Index" in middle-age and older Indonesian population: scaling exponents..., Cheung [/bib_ref] , although the predictive power was no better than WC or BMI. The aim of this study is to evaluate the power of ABSI for predicting the development of diabetes, hypertension, and dyslipidemia compared with BMI and WC in Japanese adults. This large retrospective cohort study is the first report evaluating ABSI in a Japanese population. # Materials and methods ## Study population We examined the predictive power of ABSI for development of diabetes, hypertension, and dyslipidemia by retrospective analysis of annual health examination data collected from members of Chiba City National Health Insurance for the period 2008 to 2012. The patient selection process is shown in [fig_ref] Fig 1: Selection of the subjects in this study for examining the incidence of... [/fig_ref] In 2008, 48,593 members (18,721 men, 29,872 women) age 40−70 years attended annual health examinations at designated clinics or hospitals in the city. We excluded 166 participants (77 men, 89 women) with missing information at baseline. For the analysis of incidence of diabetes, hypertension, and dyslipidemia during follow-up, 4,408 participants (2,483 men, 1,925 women) with diabetes, 21,169 (9,402 men and 11,767 women) with hypertension, and 23,746 participants (8,003 men, 15,743 women) with dyslipidemia at baseline were excluded. Diabetes was defined as fasting plasma glucose 7.0 mmol/L, hemoglobin A1c (HbA1c) 6.5%, or current use of medications for diabetes as indicated by a self-report questionnaire in accordance with guidelines of the Japanese Diabetes Society [bib_ref] The Committee of the Japan Diabetes Society on the diagnostic criteria of..., Seino [/bib_ref]. Blood samples were drawn for measurement of plasma glucose during fasting and for measurement of HbA1c in the non-fasting period. Hypertension was defined as systolic blood pressure (SBP) 140 mmHg, diastolic blood pressure (DBP) 90 mmHg, or current use of antihypertensive medications as indicated by a self-report questionnaire according to the guidelines for the management of hypertension. Dyslipidemia was defined as low-density lipoprotein cholesterol (LDL-C) 140 mg/dL, high-density lipoprotein cholesterol (HDL-C) < 40 mg/dL, or current use of medication for dyslipidemia according to the guidelines for prevention of arteriosclerosis. Furthermore, participants who did not attend annual health examinations for the period 2009 to 2012 were excluded, comprising 6,438 for diabetes analysis (2,573 men, 3,865 women), 4,168 for hypertension analysis (1,557 men, 2,611 women), and 3,905 for dyslipidemia analysis (1,683 men, 2,222 women). The final subject populations for analysis of new disease incidence during follow-up were 37,581 confirmed non-diabetics (13,588 men, 23,993 women), 23,090 confirmed non-hypertensives [fig_ref] Table 1: Baseline characteristics of all subjects divided according to the absence of disease... [/fig_ref]. Thus, we could not follow the subjects completely. There were 6,438 dropouts during follow-up in the baseline non-diabetic cohort (14.6% of the total), 4,168 (15.3%) in the baseline non-hypertensive cohort, and 3,905 (15.8%) in the baseline non-dyslipidemic cohort (S1 . There were marked differences in baseline characteristics between these dropouts and retained study subjects. For example, the dropouts were younger, more likely to be smokers, heavier, and had lower mean ABSI values compared with retained subjects in all three subgroups. In addition, dropouts had higher BMIs than the baseline non-diabetic and non-hypertensive subgroups. # Ethics statement Consent was not obtained from subjects because this study was performed using the data obtained from annual health examinations and conducted in accordance with the Act on Assurance of Medical Care for the Elderly enforced by the Ministry of Health, Labour and Welfare. To ensure anonymity, personal identifiers (e.g., name, address, and telephone number) were removed from the records, date of birth was converted into the 1st of each month, and personal ID was converted into a random number at Chiba City Hall prior to release for analysis. The Institutional Review Board of Chiba University Graduate School of Medicine approved this study. All procedures were in accordance with the Ethical Guidelines for Epidemiological Research of the Japanese Ministry of Education, Culture, Sports, Science and Technology and the Japanese Ministry of Health, Labour and Welfare. The study was conducted in accordance with the Declaration of Helsinki. ## Baseline examination We defined the results from the 2008 annual health examinations as the baseline data. Height, weight, WC, SBP, and DBP were measured and blood samples drawn during periods of fasting or non-fasting. Weight and height were measured without shoes and while wearing light clothes. WC was measured at the level of the umbilicus with an anthropometric measuring tape. However, when the umbilicus level was drooping due to excess abdominal fat accumulation, WC was measured at the midpoint between the lower border of the rib cage and the iliac crest. BMI was calculated as weight in kilograms divided by the square of height in meters. ABSI was calculated as WC/(BMI 2/3 height 1/2 ), with WC and height in meters [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref]. Serum glucose, HbA1c, LDL-C, HDL-C, AST, ALT, and GGT were determined by commercial clinical laboratories. Subjects were categorized according to the following parameters. (1) Diabetes category (normal, borderline, and diabetic) was determined on the basis of baseline fasting plasma glucose, HbA1c, or self-reported use of medications for diabetes according to the guidelines of the Japanese Diabetes Society [bib_ref] The Committee of the Japan Diabetes Society on the diagnostic criteria of..., Seino [/bib_ref] ; with normal (no diabetes) defined as fasting plasma glucose < 6.1 mmol/L or HbA1c < 6.0% and no history of diabetes medication use; borderline as fasting plasma glucose between 6.1 and < 7.0 mmol/L or HbA1c between 6.0% and < 6.5% and no use of medication for diabetes; and diabetic as fasting plasma glucose 7.0 mmol/L, or HbA1c 6.5% or current use of medication for diabetes. [bib_ref] Association of all-cause mortality with overweight and obesity using standard body mass..., Flegal [/bib_ref] Smoking status (non-smoker, smoker) was defined using a self-report questionnaire. Plasma concentrations of HDL-C, AST, ALT, and GGT were transformed logarithmically because the distributions of these variables were not normal. ## Incidence of diabetes, hypertension, and dyslipidemia Results of annual health examinations from 2009 to 2012 were used as the follow-up data. Each follow-up examination included routine blood analysis, blood pressure measurement, and completion of a self-report questionnaire about medications for diabetes, hypertension, and dyslipidemia. Fasting plasma glucose (or HbA1c), LDL-C, and HDL-C were measured at each follow-up examination. Subjects with fasting plasma glucose 7.0 mmol/L, HbA1c 6.5%, or reporting medication for diabetes during follow-up but without diabetes at baseline were defined as new diabetic patients. Similarly, subjects with SBP 140 mmHg, DBP 90 mmHg, or reporting medication for hypertension during follow-up but without hypertension at baseline were defined as new hypertensive patients. Subjects with LDL-C 140 mg/dL, HDL-C < 40 mg/dL, or reporting medication for dyslipidemia but without dyslipidemia at baseline were defined as new dyslipidemic patients. # Statistical analysis Continuous variables are presented as mean ± standard deviation. For each disease, comparison of variables between cases and controls was performed using unpaired t-tests or chi-square tests as appropriate and in case-matched subgroups using McNemar's test or the paired t-test as appropriate. We also calculated the standardized difference (SD) between cases and controls for all subjects and for case-matched subgroups. The correlations among BMI, WC, and ABSI were evaluated by Pearson's correlation coefficients. Calculating Z-scores. For standardization, Z-scores for height, weight, BMI, WC, and ABSI were calculated as reported by Krakauer et al. [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref]. First, we computed the sample mean and standard deviation for each age (in years) separately for all men and women (48,759 subjects in total) who did not have missing data at baseline (each marker in . Second, we obtained the predicted mean and standard deviation for each age from the quadratic curve that described the change in mean or standard deviation with age (trend lines in . From measurement values, predicted means, and standard deviations, Z-scores were calculated as follows: (measurement value-predicted mean)/predicted standard deviation. Logistic regression analysis. We conducted logistic regression analysis to examine the associations of standardized BMI, WC, and ABSI (Z-scores) with incidences of diabetes, hypertension, and dyslipidemia. Logistic regression analyses were performed using two model types: sex-and age-adjusted models and multivariable-adjusted models including the covariates of sex, age, diabetes category (normal, borderline, or diabetes), SBP, DBP, LDL-C, log [HDL-C], log [AST], log [ALT], log [GGT], and smoking habit at baseline depending on application. All factors were included in the model for new diabetes incidence in the baseline non-diabetic cohort except for diabetes category; all except SBP and DBP for analysis of new hypertension incidence in the baseline non-hypertensive cohort; and all except LDL-C and log [HDL-C] for analysis of new dyslipidemia incidence in the baseline non-dyslipidemic cohort. Each regression was conducted both with and without BMI adjustment. For sensitivity analysis, logistic regression was also performed in subgroups stratified by sex. The model selection criteria were Akaike's information criterion (AIC) and area under the curve (AUC) from receiver operating characteristic analysis. Case matching. As shown in [fig_ref] Table 1: Baseline characteristics of all subjects divided according to the absence of disease... [/fig_ref] , there were significant differences in baseline parameters and a large patient number discrepancy between cases (subjects who would eventually be diagnosed) and controls (those who would not be diagnosed with diabetes, hypertension, or dyslipidemia during follow-up). Therefore, case selection was performed by employing the propensity score matching method with a greedy 5-to-1 digit-matching algorithmfor control of baseline characteristics. Variables used for calculating propensity scores were sex, age, diabetes category, smoking habit, SBP, DBP, LDL-C, log [HDL-C], log [aspartate aminotransferase, AST], log [alanine aminotransferase, ALT], and log [gamma-glutamyltransferase, GGT] depending on application. That is, all variables except diabetes category were included for the analysis of new diabetes incidence; all except SBP and DBP for analysis of new hypertension incidence; and all except LDL-C and log [HDL-C] for analysis of new dyslipidemia incidence. The greedy 5-to-1 digit-matching algorithm yields "best" matches first and then "next-best" matches in a hierarchical sequence until no more matches can be made. Greedy 5-to-1 digit- [formula] Fig 2. [/formula] Mean and standard deviation of ABSI, BMI, and WC stratified by sex and age. Abbreviations: BMI, body mass index; WC, waist circumference; ABSI, a body shape index. Each marker shows mean and standard deviation stratified by sex and age. Black squares, men; gray circles, women. matching means that the cases were first matched to controls on 5 digits of the propensity score. For those that did not match, cases were then matched to controls on 4 digits of the propensity score. This continued down to a 1-digit match on propensity score for subjects that remained unmatched. This algorithm yields 1:1 case−control pairs. After this process, there were 1,947 pairs for diabetes, 6,962 pairs for hypertension, and 7,263 pairs for dyslipidemia. Conditional logistic regression models were used to assess the associations of BMI, WC, and ABSI with each disease in case-matched subgroups yielded by the propensity score matching method. All comparisons were planned and all tests two-tailed. A p-value < 0.05 was considered statistically significant. All statistical analyses were performed using SAS software version 9.4 (SAS Institute, Cary, NC) and STATA13 software package (Stata Corp., College Station, TX). # Results The mean ABSI for the entire cohort increased with age in both men and women . Means were similar in younger men and women (< 55 years) but the increase with age was larger in women thereafter and ABSI was more variable in women at all ages . In contrast, mean BMI and WC exhibited modest age-dependent decreases in men (larger for BMI), while women exhibited age-dependent increases with similar trajectories. The SDs of BMI and WC were similar in men and women, with a nearly parallel age-dependent decrease . The standardized WC was strongly correlated with weight (r = 0.803) and with BMI (r = 0.809), while ABSI did not correlate with weight (r = −0.006) or BMI (r = −0.034) and the correlation with WC was only moderate (r = 0.530). The baseline characteristics of all subjects (data from 2008), with and without eventual development of the disease of interest during follow-up (2009−2012), are shown in [fig_ref] Table 1: Baseline characteristics of all subjects divided according to the absence of disease... [/fig_ref]. Almost all baseline characteristics differed between the case group and control group. In total, 1,947 of 37,581 subjects confirmed to be non-diabetic at baseline were eventually diagnosed with diabetes during the follow-up period (5.2%); 6,963 of 23,090 subjects confirmed to be non-hypertensive at baseline were eventually diagnosed with hypertension (30.2%); and 7,263 of 20,776 subjects without dyslipidemia at baseline were eventually diagnosed with dyslipidemia (35.0%). For all three diseases, BMI, WC, and ABSI at baseline were significantly larger in the case group than the control group (p < 0.001). [fig_ref] Table 2: Associations between disease incidence [/fig_ref] shows the odds ratios (ORs) of standardized BMI, WC, and ABSI values (Z-scores) for development of diabetes, hypertension, and dyslipidemia in age-and sex-adjusted models. ## Logistic regression analyses For diabetes without BMI adjustment, all three parameters (BMI, WC, and ABSI) were positively associated with disease incidence (p < 0.001). However, the larger AIC and significantly smaller AUC in the model with ABSI compared with models with BMI (p < 0.001) or WC (p < 0.001) indicate that the predictive power of ABSI is weaker than those of BMI or WC for diabetes. With BMI adjustment, the predictive powers of WC and ABSI remained significant (both p < 0.001). The result of smaller AIC and significantly greater AUC in the model with ABSI than that with WC (p = 0.039) suggests that the predictive power of ABSI independent of BMI for diabetes is greater than that of WC. However, the observation that AUCs was not significantly different (p = 0.180) with ABSI and WC in the multivariable-adjusted models [fig_ref] Table 3: Associations between new disease incidence [/fig_ref] indicates that both parameters have similar predictive powers. For hypertension without BMI adjustment, elevated BMI and WC were significantly associated with increased disease incidence (p < 0.001), while ABSI was not (p = 0.188), as shown in [fig_ref] Table 2: Associations between disease incidence [/fig_ref]. The greater AIC was observed in the model with ABSI than those with BMI and WC, while a significantly smaller AUC was observed in the ABSI model compared with those with BMI and WC (both p < 0.001). With BMI adjustment, neither WC nor ABSI was significantly associated with the incidence of hypertension (p = 0.273 and p = 0.079, respectively). The AICs were similar in models with WC or ABSI, and the AUCs did not differ significantly (p = 0.383). Thus, similar to diabetes, the predictive power of ABSI for hypertension incidence was weaker than those of BMI and WC when BMI was not adjusted, while the powers of ABSI and WC were similar after BMI adjustment. These results are consistent with those in the multivariableadjusted models [fig_ref] Table 3: Associations between new disease incidence [/fig_ref] , where BMI and WC were significant predictors of hypertension incidence (p < 0.001), while ABSI was not (p = 0.742). With BMI adjustment, neither WC nor ABSI was a significant predictor of hypertension. For dyslipidemia without BMI adjustment, all three parameters (BMI, WC, and ABSI) were positively associated with disease incidence (p < 0.001, p < 0.001, and p = 0.001, respectively) in sex-and age-adjusted models. Again, the greater AIC and significantly smaller AUC was observed in the model with ABSI compared with those with BMI or WC (both p < 0.001). After BMI adjustment, WC and ABSI were significantly associated with the incidence of dyslipidemia (both p < 0.001), showing similar AICs and AUCs (p = 0.482). These results are consistent with those for multivariable-adjusted models [fig_ref] Table 3: Associations between new disease incidence [/fig_ref] , where all three parameters were predictive of dyslipidemia without BMI adjustment (p < 0.001, p < 0.001, and p = 0.011, respectively) but the predictive power of ABSI was slightly weaker as indicated by the AIC and AUC values. Both WC and ABSI were also significant predictors in models with BMI adjustment (both p = 0.002). The ORs were also calculated and data were stratified by BMI, WC, and ABSI Z-score ranges both without and with BMI correction [fig_ref] Fig 4: Odds ratio and 95% confidence interval of WC and ABSI categories for... [/fig_ref]. Results were consistent with analysis of the continuous data [fig_ref] Table 3: Associations between new disease incidence [/fig_ref]. # Sensitivity analysis We also performed logistic regression analysis in subgroups stratified by sex [fig_ref] Table 4: Sensitivity analysis in subgroups stratified by sex [/fig_ref]. These results were generally consistent with those for the individual baseline non-diabetic, non-hypertensive, and non-dyslipidemic groups [fig_ref] Table 3: Associations between new disease incidence [/fig_ref]. However, in men, compared with ABSI, BMI and WC were not stronger predictors of diabetes when BMI was not adjusted [fig_ref] Table 4: Sensitivity analysis in subgroups stratified by sex [/fig_ref] (p for AUC = 0.999 and 0.829, respectively). Furthermore, the interactions between sex and BMI and between sex and WC were significant (p < 0.001), while that between sex and ABSI was not (p = 0.465), suggesting that the effects of high BMI and WC on diabetes incidence are greater in women than in men, but the effect of ABSI does not differ by sex. ## Study of case-matched subset by propensity score All baseline characteristics differed significantly between cases and controls for all three diseases in the total cohorts [fig_ref] Table 1: Baseline characteristics of all subjects divided according to the absence of disease... [/fig_ref] , but no significant differences remained in the variables used for calculating propensity score following propensity score matching [fig_ref] Table 5: Baseline characteristics of controls and cases after propensity score matching [/fig_ref] , and all imbalances between cases and controls were less than 20%. [fig_ref] Table 6: Associations between disease incidence [/fig_ref] shows the results of conditional logistic regression analysis for these case-matched subgroups. Results indicate that compared with WC and BMI, ABSI had weaker predictive power for the incidence of all three diseases. # Discussion Our retrospective cohort study revealed that compared with BMI and WC, ABSI was not a better predictor of diabetes, hypertension, or dyslipidemia incidence in Japanese adults. In addition, using propensity score matching as an alternative evaluation method, the predictive power of ABSI for all three diseases was also lower than those of BMI and WC. In American Caucasians, high ABSI accounted for a greater proportion of the total mortality hazard than did elevated BMI [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref]. There are two possible reasons for this discrepancy. One is the different outcome measures used: all-cause mortality versus specific disease conditions. The other is racial differences. In regard to the first reason, a U-shaped or J-shaped association between all-cause mortality and BMI has been reported [bib_ref] Bodymass index and mortality among 1.46 million white adults, Berrington De Gonzalez [/bib_ref] [bib_ref] Body mass index, waist circumference and waist-hip ratio: which is the better..., Czernichow [/bib_ref] [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref] , while the associations of diabetes, hypertension, and dyslipidemia incidence [bib_ref] Obesity, weight change and risks for hypertension, diabetes and hypercholesterolemia in Japanese..., Ishikawa-Takata [/bib_ref] and prevalence [bib_ref] The relationship of body mass index to diabetes mellitus, hypertension and dyslipidaemia:..., Bays [/bib_ref] [bib_ref] Associations between body mass index and development of metabolic disorders in fertile..., Schmiegelow [/bib_ref] with BMI are typically linear or nearly linear. In our study, near-linear associations between BMI and the incidence of diabetes and hypertension were also observed. In regard to the second reason, morphometric and physiological racial differences may alter the health implications of these indices. For example, the prevalence of obesity in Japanese adults was much lower than Americans. In addition, East Asians, including the Japanese, are known to have limited innate capacity for insulin secretion, resulting in greater susceptibility to type 2 diabetes than Caucasians [bib_ref] Ethnic differences in the relationship between insulin sensitivity and insulin response: a..., Kodama [/bib_ref]. A prospective cohort study of Chinese adults of a smaller scale (n = 687) with the incidence of diabetes as the primary outcome has reported that the predictive power of ABSI was no better than that of WC or BMI [bib_ref] Could the new body shape index predict the new onset of diabetes..., He [/bib_ref]. In another study, the association of ABSI with hypertension incidence was found to be weaker than WC or BMI among 8,255 Indonesians [bib_ref] A Body Shape Index" in middle-age and older Indonesian population: scaling exponents..., Cheung [/bib_ref]. Our results were consistent with those of these two studies of Asian subjects that examined the incidence of diabetes or hypertension as outcomes, even though the precise reasons for the discrepancy were unable to be ascertained. It should be noted that correlations between BMI, WC, and ABSI in our study were similar to those of Krakauer et al [bib_ref] A new body shape index predicts mortality hazard independently of body mass..., Krakauer [/bib_ref]. In brief, both studies found a high correlation between BMI and WC, a moderate correlation between ABSI and WC, but almost no correlation between ABSI and BMI. ABSI was significantly associated with the incidence of diabetes and dyslipidemia even after adjustment for BMI. These BMI-independent associations may reflect the fact that ABSI represents central body mass, such as higher visceral fat accumulation, higher trunk fat accumulation, and lesser limb lean mass. Furthermore, in the sex-and age-adjusted models, the finding that the predictive power of ABSI for diabetes independent of BMI was significantly greater than that of WC, could be interesting [fig_ref] Table 2: Associations between disease incidence [/fig_ref]. If this is true, ABSI has an advantage over WC in predicting diabetes. However, this association was not observed in the multivariable-adjusted models [fig_ref] Table 3: Associations between new disease incidence [/fig_ref] , after propensity score matching [fig_ref] Table 6: Associations between disease incidence [/fig_ref] , or in sensitivity analyses [fig_ref] Table 4: Sensitivity analysis in subgroups stratified by sex [/fig_ref]. Thus, the stronger predictive power of ABSI might be confounded by other co-varying baseline characteristics. [aspartate aminotransferase], log [alanine aminotransferase], log [gamma-glutamyltransferase] and smoking habit, depending on application. Diabetes category was excluded for the analysis of diabetes, systolic blood pressure and diastolic blood pressure for the analysis of hypertension, and low-density lipoprotein cholesterol and log [high-density lipoprotein cholesterol] for the analysis of dyslipidemia. doi:10.1371/journal.pone.0128972.g003 We found that the predictive powers of BMI and WC for diabetes incidence are greater in women than in men, while that of ABSI does not differ by sex [fig_ref] Table 4: Sensitivity analysis in subgroups stratified by sex [/fig_ref]. Cross-sectional studies in the Japanese population [bib_ref] Impacts of visceral adipose tissue and subcutaneous adipose tissue on metabolic risk..., Oka [/bib_ref] and in the Framingham Heart Study [bib_ref] Abdominal visceral and subcutaneous adipose tissue compartments: association with metabolic risk factors..., Fox [/bib_ref] have shown that associations of metabolic risk factors, such as systolic and diastolic blood pressure, fasting glucose, and total cholesterol, with increasing volumes both of subcutaneous and visceral fat, strictly measured by computed tomography, were stronger in women than in men. Our observation of a sex difference in BMI and WC could be the same phenomenon, but this is not the case with ABSI. Thus, ABSI might have an implication for disease incidence that is different from BMI or WC. Further studies are necessary to conclude whether this difference is true or not. The following four limitations in our study are conceivable. First, due to the retrospective cohort design, we could not obtain other potentially relevant data, such as alcohol consumption and levels of physical activity, which may confound the associations of BMI, WC, and ABSI with disease incidence. To correct this, we have made adjustment for potential confounding factors as much as possible using logistic regression analysis and the propensity score matching method. Second, fasting blood glucose data for some subjects were not available. To compensate for this, we employed HbA1c data and history of diabetes medication to obtain an accurate diagnosis. Third, some bias might exist that resulted in underestimation of associations because the reasons and outcomes of non-attendance of annual health checkup are unknown. Fourth, the generalizability of our findings might be limited because the subjects are mainly elderly adults living in one urban area of Japan with a relatively higher incidence of hypertension (30.2%) and dyslipidemia (35.0%) during the 4-year period. On the other hand, the major advantage of our study is that we obtained consistent results using two different covariate adjustment methods (logistic regression analysis and the propensity score matching) and also from sensitivity analyses. Furthermore, new-onset diabetes, hypertension, and dyslipidemia was confirmed both by self-reported medication use and physiological measures. Since subjects are not always aware of their disease, we may underestimate disease incidence when it is determined only by self-reported medication use. In conclusion, ABSI was not a better predictor of diabetes, hypertension, and dyslipidemia incidence than BMI and WC in Japanese adults. However, ABSI had significant associations with the incidence of diabetes and dyslipidemia independently of BMI. Supporting Information S1 Baseline characteristics of retained subjects and dropouts during follow-up. (DOCX) [fig] Fig 1: Selection of the subjects in this study for examining the incidence of diabetes, hypertension, and dyslipidemia.doi:10.1371/journal.pone.0128972.g001 [/fig] [fig] Fig 4: Odds ratio and 95% confidence interval of WC and ABSI categories for development of diabetes, hypertension and dyslipidemia with BMI adjustment. Abbreviations: BMI, body mass index; WC, waist circumference; ABSI, a body shape index; AIC, Akaike's information criterion; AUC, area under the curve; 95% CI, 95% confidence interval. Adjusted for BMI category in addition to same variables as shown inFig 3. [/fig] [table] Table 1: Baseline characteristics of all subjects divided according to the absence of disease at baseline (diabetes, hypertension, dyslipidemia) and subdivided according to onset of new disease during follow-up (cases vs. controls).ABSI, a body shape index; WC, waist circumference; BMI, body mass index; SD, standardized difference; SBP, systolic blood pressure; DBP, diastolic blood pressure; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyltransferase a Percentage and p-value by the chi-square test b Mean (standard deviation) and p-value by the unpaired t-test doi:10.1371/journal.pone.0128972.t001 [/table] [table] Table 2: Associations between disease incidence (diabetes, hypertension, dyslipidemia) and Z-scores for BMI, WC, and ABSI in sex-and age-adjusted models. [/table] [table] Table 3: Associations between new disease incidence (diabetes, hypertension, and dyslipidemia) and Z-scores for BMI, WC, and ABSI as revealed by logistic regression analysis of all subjects in the multivariable-adjusted model. pressure, low-density lipoprotein cholesterol, log [high-density lipoprotein cholesterol], log [aspartate aminotransferase], log [alanine aminotransferase], log [gamma-glutamyltransferase] and smoking habit, depending on application. The diabetes category was omitted for the analysis of new diabetes incidence, systolic blood pressure and diastolic blood pressure were omitted for analysis of new hypertension incidence, and low-density lipoprotein cholesterol and log [high-density lipoprotein cholesterol] were omitted for the analysis of new dyslipidemia incidence.3 Adjusted for BMI Z-score and the variables described above [/table] [table] Table 4: Sensitivity analysis in subgroups stratified by sex. [/table] [table] Table 5: Baseline characteristics of controls and cases after propensity score matching. SD, standardized difference; SBP, systolic blood pressure; DBP, diastolic blood pressure; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density lipoprotein cholesterol; AST, aspartate aminotransferase; ALT, alanine aminotransferase; GGT, gamma-glutamyltransferase a Percentage and p-value by McNemar's test b Mean (standard deviation) and p-value by paired t-test c These variables were used for calculating the propensity score doi:10.1371/journal.pone.0128972.t005 [/table] [table] Table 6: Associations between disease incidence (diabetes, hypertension, and dyslipidemia) and Z-scores for BMI, WC, and ABSI as revealed by conditional logistic regression analysis of case-matched subsets obtained by propensity score matching.OR, odds ratio; CI, confidence interval; ABSI, a body shape index; WC, waist circumference; BMI, body mass index; AIC, Akaike's information criterion; AUC, area under the curve 1 p-value with Bonferroni's correction 2 Adjusted for BMI doi:10.1371/journal.pone.0128972.t006 Conceived and designed the experiments: MF YS KN. Performed the experiments: MF AH. Analyzed the data: MF. Contributed reagents/materials/analysis tools: YS KN ST. Wrote the paper: MF YS KN ST AH. Data acquisition: MF AH. Data interpretation: YS KN ST. Supervision or mentorship: AH. [/table]

UVR8-mediated inhibition of shade avoidance involves HFR1 stabilization in Arabidopsis ## S1 fig

Time‐resolved interaction proteomics of the GIGANTEA protein under diurnal cycles in Arabidopsis The plant-specific protein GIGANTEA (GI) controls many developmental and physiological processes, mediating rhythmic post-translational regulation. GI physically binds several proteins implicated in the circadian clock, photoperiodic flowering, and abiotic stress responses. To understand GI's multifaceted function, we aimed to comprehensively and quantitatively identify potential interactors of GI in a time-specific manner, using proteomics on Arabidopsis plants expressing epitope-tagged GI. We detected previously identified (in)direct interactors of GI, as well as proteins implicated in protein folding, or degradation, and a previously uncharacterized transcription factor, CYCLING DOF FACTOR6 (CDF6 ). We verified CDF6's direct interaction with GI, and ZEITLUPE/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1/LIGHT KELCH PROTEIN 2 proteins, and demonstrated its involvement in photoperiodic flowering. Extending interaction proteomics to time series provides a data resource of candidate protein targets for GI's post-translational control. Arabidopsis thaliana plants have well-documented 24-h rhythms in many physiological processes, from hypocotyl elongation to photosynthetic functions as well as defense responses against pathogen and herbivore attack [bib_ref] The circadian regulation of photosynthesis, Dodd [/bib_ref] [bib_ref] The intracellular dynamics of circadian clocks reach for the light of ecology..., Millar [/bib_ref]. The overt circadian rhythms are driven by intricate transcriptional-translational feedback loops [bib_ref] The intracellular dynamics of circadian clocks reach for the light of ecology..., Millar [/bib_ref]. Detailed dynamic models based mostly upon transcriptional repression recapitulate the rhythmic expression profiles of these clock genes, including manipulations of the system in mutant plants and under changing photoperiods [bib_ref] Defining the robust behaviour of the plant clock gene circuit with absolute..., Flis [/bib_ref] [bib_ref] Photoperioddependent changes in the phase of core clock transcripts and global transcriptional..., Flis [/bib_ref] [bib_ref] The clock gene circuit in Arabidopsis includes a repressilator with additional feedback..., Pokhilko [/bib_ref] [bib_ref] Rethinking transcriptional activation in the Arabidopsis circadian clock, Fogelmark [/bib_ref]. Gene expression switches can operate on a timescale of minutes. However, it does not obviously explain the slow 24-h timescale of circadian clocks. In addition to chromatin modification [bib_ref] The impact of chromatin dynamics on plant light responses and circadian clock..., Barneche [/bib_ref] , the slow degradation rate of the transcriptional repressors extends the timescale of transcriptional regulation, which mathematical analysis has long identified and experiments have confirmed [bib_ref] Dependence of the period on the rate of protein degradation in minimal..., Gerard [/bib_ref] [bib_ref] The relationship between FRQ-protein stability and temperature compensation in the Neurospora circadian..., Ruoff [/bib_ref]. In Arabidopsis, regulated protein degradation is also crucial to circadian timing and provides one mechanism for environmental light signals to control the pace of the clock. One of the central proteins that regulate the degradation rate of circadian clock proteins in the Arabidopsis clock is the GIGANTEA (GI) protein. The gi mutants were originally identified as delayed-flowering mutants under long-day conditions where wild-type plants flower early [bib_ref] Supervital mutants of Arabidopsis, R Edei [/bib_ref] [bib_ref] A genetic and physiological analysis of late flowering mutants in Arabidopsis thaliana, Koorneef [/bib_ref]. The gi mutants also alter the pace of the circadian clock [bib_ref] Control of circadian rhythms and photoperiodic flowering by the Arabidopsis GIGANTEA gene, Park [/bib_ref] [bib_ref] GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and..., Fowler [/bib_ref] [bib_ref] Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in..., Mizoguchi [/bib_ref] [bib_ref] Forward genetic analysis of the circadian clock separates the multiple functions of..., Kevei [/bib_ref] [bib_ref] GIGANTEA acts in blue light signaling and has biochemically separable roles in..., Martin-Tryon [/bib_ref]. GI affects the clock through interaction with the F-box proteins of the ZEITLUPE (ZTL)/FLAVIN-BINDING, KELCH REPEAT, F-BOX 1 (FKF1)/LIGHT KELCH PRO-TEIN 2 (LKP2) family, and increases the degradation of the evening-expressed circadian repressors, TIMING OF CAB EXPRESSION 1 (TOC1), and PSEUDO RESPONSE REGULATOR 5 (PRR5) [bib_ref] ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light, Kim [/bib_ref] [bib_ref] Targeted degradation of PSEUDO-RESPONSE REGULATOR5 by an SCF ZTL complex regulates clock..., Kiba [/bib_ref] [bib_ref] Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana, Mas [/bib_ref]. The degradation is directly mediated by the ZTL/FKF1/ LKP2 proteins, together with ARABIDOPSIS SKP1-LIKE (ASK) and CULLIN (CUL) proteins, to form an SKP-CUL-F-box (SCF) ubiquitin ligase complex that targets these clock proteins to the proteasome [bib_ref] Targeted degradation of PSEUDO-RESPONSE REGULATOR5 by an SCF ZTL complex regulates clock..., Kiba [/bib_ref] [bib_ref] Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana, Mas [/bib_ref] [bib_ref] Formation of an SCF ZTL complex is required for proper regulation of..., Han [/bib_ref] [bib_ref] F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control..., Baudry [/bib_ref]. ZTL binds to GI in a light-dependent manner [bib_ref] ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light, Kim [/bib_ref]. This interaction stabilizes both ZTL and GI. ZTL is thought to not only enhance GI stability but also to sequester GI in the cytoplasm. GI is rhythmically expressed due to circadian control of GI transcription, and therefore GI confers rhythmicity upon ZTL protein levels [bib_ref] The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA, Kim [/bib_ref] [bib_ref] ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration, Kim [/bib_ref] [bib_ref] Balanced nucleocytosolic partitioning defines a spatial network to coordinate circadian physiology in..., Kim [/bib_ref]. GI mRNA levels peak 8-10 h after dawn [bib_ref] GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and..., Fowler [/bib_ref] [bib_ref] Temporal repression of core circadian genes is mediated through EARLY FLOWERING 3..., Dixon [/bib_ref] [bib_ref] Arabidopsis GIGANTEA protein is posttranscriptionally regulated by light and dark, David [/bib_ref] , before repression by the evening complex, which are composed of EARLY FLOWERING 3 (ELF3), ELF4, and LUX ARRHYTHMO (LUX, also termed PHYTOCLOCK1) [bib_ref] Temporal repression of core circadian genes is mediated through EARLY FLOWERING 3..., Dixon [/bib_ref] [bib_ref] The ELF4-ELF3-LUX complex links the circadian clock to diurnal control of hypocotyl..., Nusinow [/bib_ref]. GI protein interacts with and is destabilized by ELF3 and COP1 [bib_ref] COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI..., Yu [/bib_ref]. Within the nucleus, the clock protein ELF4 interacts with GI and sequesters it away from the promoter of the floral induction gene CONSTANS (CO), contributing to rhythmic regulation of CO [bib_ref] ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration, Kim [/bib_ref]. The rhythm of CO expression provides part of the timing function required to distinguish long days from short days. CO protein activates transcription of FLOWERING LOCUS T (FT) [bib_ref] Distinct roles of CONSTANS target genes in reproductive development of Arabidopsis, Samach [/bib_ref]. GI physically associates with the promoter regions of CO and FT [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] [bib_ref] GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana, Sawa [/bib_ref] , and also binds to transcriptional regulators of CO [bib_ref] TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis, Kubota [/bib_ref]. Morning-expressed CYCLING DOF FAC-TOR 1 (CDF1), CDF2, CDF3, and CDF5 directly repress CO and FT transcription, delaying flowering in long days [bib_ref] FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in..., Imaizumi [/bib_ref] [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref] [bib_ref] FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering, Song [/bib_ref]. The F-box protein FKF1 is co-expressed with GI, binds both to GI and to CDF1-CDF5 under light conditions, and initiates the degradation of CDFs by ubiquitination [bib_ref] FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in..., Imaizumi [/bib_ref] [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref]. Thus, GI facilitates expression of CO and FT at the end of long days, by relieving CDF repression [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref] [bib_ref] FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering, Song [/bib_ref]. In addition to its roles in the clock and flowering, GI has been linked to carbon metabolism, [bib_ref] Monogenic recessive mutations causing both late floral initiation and excess starch accumulation..., Eimert [/bib_ref] [bib_ref] Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf..., Mugford [/bib_ref] [bib_ref] The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis..., Dalchau [/bib_ref] and various stress responses. GI confers tolerance to high salinity through interaction with the protein kinase SALT OVERLY SENSITIVE 2 (SOS2) [bib_ref] Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in..., Kim [/bib_ref] and is involved in ELF under drought conditions [bib_ref] GIGANTEA enables drought escape response via abscisic acid-dependent activation of the florigens..., Riboni [/bib_ref]. Moreover, mutations in GI increase resistance to oxidative stress [bib_ref] Oxidative stress tolerance and longevity in Arabidopsis: the late-flowering mutant gigantea is..., Kurepa [/bib_ref] and freezing [bib_ref] Involvement of GIGANTEA gene in the regulation of the cold stress response..., Cao [/bib_ref] due to increased CDF expression levels [bib_ref] The GI-CDF module of Arabidopsis affects freezing tolerance and growth as well..., Fornara [/bib_ref]. GI's biochemical mechanisms in most of these responses are unknown. GIGANTEA's role in the clock is mediated at the biochemical level by co-chaperone activity, which involves binding to HEAT SHOCK PROTEIN 90 (HSP90) and appears to stabilize ZTL [bib_ref] HSP90 functions in the circadian clock through stabilization of the client F-box..., Kim [/bib_ref] [bib_ref] GIGANTEA is a cochaperone which facilitates maturation of ZEITLUPE in the Arabidopsis..., Cha [/bib_ref]. This activity can affect other test substrates but its other native targets, if any, are unknown. As outlined above, GI's known functions with the ZTL family and SOS2 are mediated by protein-protein interactions. Therefore, GI has been suggested to serve as a scaffold or hub protein that orchestrates other protein interactions [bib_ref] Photoperiodic control of flowering: not only by coincidence, Imaizumi [/bib_ref] , for example to provide chaperone activity [bib_ref] GIGANTEA is a cochaperone which facilitates maturation of ZEITLUPE in the Arabidopsis..., Cha [/bib_ref]. Although such protein interactions are thought to mediate GI's functions, these interactions have not been comprehensively and quantitatively analyzed. We therefore conducted interaction proteomics assays using the GI protein, and obtained time-resolved data on potential direct and indirect partners of GI, over the daily time course. Here, we discuss the abundance profiles of proteins co-immunoprecipitated with GI, and functions of new candidate interactors and highlight a DOF protein, which we refer to as CDF6, validating its direct interactions and functional importance. # Materials and methods ## Generation of plant materials To generate plants with epitope-tagged GI protein, gi-2 mutants were transformed with a construct expressing C-terminal 3xFLAG-6His-tagged GI protein (GI-3F6H). The full-length GI cDNA without the stop codon was amplified and inserted into pENTR/D-TOPO vector (Invitrogen, Carlsbad, CA, USA). After sequence verification, the GI cDNA was transferred into the pB7HFC vector, designed for in-frame epitope fusion [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] by a Gateway cloning reaction (Invitrogen). pB7HFC-GI-3F6H was introduced to the gi-2 mutant by Agrobacterium-mediated transformation. Transgenic plants that rescued the gi-2 phenotype were selected, and the expression of the GI-3F6H protein was verified by western blotting (as in [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] ; Methods S1). Samples for the preliminary and qualitative GI tandem affinity purification (TAP)-mass spectrometry (MS) studies were prepared as described in [bib_ref] Distinct roles of FKF1, GIGANTEA, and ZEITLUPE proteins in the regulation of..., Song [/bib_ref]. To generate SUC2:HA-CDF6 plants, the CDF6 CDS (AT1G26790) was PCR-amplified using cDNA derived from 2-week-old long-day grown plants as a template, and cloned into pENTR D-TOPO (Invitrogen), to form pENTR HA-CDF6. 2.3 kbp of the SUC2 5 0 upstream promoter region was amplified and cloned into the pENTR 5 0 -TOPO vector (Invitrogen), to form pENTR 5 0 SUC2. Using a sequential LR clonase II reaction (Invitrogen), we integrated the pENTR 5' SUC2, pENTR HA-CDF6 into the R4pGWB501 vector [bib_ref] Development of R4 gateway binary vectors (R4pGWB) enabling high-throughput promoter swapping for..., Nakagawa [/bib_ref] , to form SUC2:HA-CDF6. After confirming the sequence, this vector was transformed into Col-0 WT plants using by Agrobacterium-mediated transformation. Transgenic plants were selected based on the expression level of CDF6 transcript. [fig_ref] Figure 2: GI-TAP time series [/fig_ref] and flash-frozen in liquid nitrogen. Dim green safelight was used to harvest samples during darkness. The same total number of WT control samples were harvested as GI-3F6H replicates at each time point, spread out across the time series (leaving out ZT15 and ZT31). ## Plant growth conditions ## Quantitative pcr (qpcr) analysis Seedlings were ground into powder with a mortar and pestle with liquid nitrogen, and total RNA was isolated by using an illustra RNAspin Mini kit (GE Healthcare, Chicago, IL, USA) according to the manufacturer's instructions. Two microgram of total RNA was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad, Hercules, CA, USA) according to the manufacturer's instructions. cDNA was diluted five times with water, and 2 lL was used as a template for quantitative PCR (qPCR) analysis using primers as shown in . ISOPENTE-NYL PYROPHOSPHATE/DIMETHYLALLYL PYROPH-OSPHATE ISOMERASE 2 (IPP2) was used as an internal control for normalization. The average value from WT was set to 1.0 to calculate the relative expression of other lines. To amplify CO and CDF6, three-step PCR cycling program was used: 1 min at 95°C, followed by 40-50 cycles of 10 s at 95°C, melting temperatures for 15 or 20 s, and 72°C extension for 15 s. To amplify GI, FT, and IPP2, a two-step PCR cycling program was used: 1 min at 95°C, followed by 40-50 cycles of 10 s at 95°C and 20 s at 60°C. Data show the average of three biological replicates with SEM; each measurement had two technical replicates. ## Protein extraction and tandem affinity purification (tap) procedure All steps in the protein extraction, TAP, and preparation for MS were carried out in random sample order to avoid bias due to order of processing. Frozen plant tissue was ground to a fine powder in a liquid nitrogen and dry icecooled mortar and processed essentially as described [bib_ref] Distinct roles of FKF1, GIGANTEA, and ZEITLUPE proteins in the regulation of..., Song [/bib_ref]. Detailed procedures are described in Supporting Experimental Procedures (Methods S2). ## Protein digestion and mass spectrometric analysis Preparation of samples for MS for the qualitative and time series studies analysis used an on-bead digest, prior to mass spectrometric analysis. Detailed procedures are described in Supporting Experimental Procedures (Methods S3). ## Proteomics data analysis and bioinformatics For the qualitative study, database searches were performed using Comet [bib_ref] Comet: an open-source MS/MS sequence database search tool, Eng [/bib_ref] , searching against the Uniprot Arabidopsis protein sequence database, and using Percolator (Matrix Science, Boston, MA, USA) with a q-value cutoff of 0.01. Cysteine residue masses were considered statically modified by iodoacetamide, and methionine dynamically modified by a single oxidation. Precursor mass tolerance was 10 p.p.m., and product ion tolerance was 0.5 Da. The principle of parsimony was used for protein inference, and at least two unique peptides were required for each identified protein. The time series data were analyzed using the commercial Progenesis LC-MS software (version 4.1.4924.40586; Nonlinear Dynamics, Newcastle, UK) for label-free quantitation. Raw files were imported into a label-free analysis experiment, chromatograms were subjected to automatic alignment and peak picking. Only charges 2+, 3+, and 4+ and data from 25 to 75 min of the runs were chosen for analysis. The exported file of MS/MS spectra was uploaded on the Mascot website (version 2.4) and a search was carried out with the following parameters: database Arabidopsis_1rep (version 20110103), trypsin as enzyme, allowing up to two missed cleavages, carbamidomethyl (C) as a fixed modification, Oxidation (M), Phospho (ST) and Phospho (Y), as variable modifications, a peptide tolerance of 10 p.p.m., and MS/MS tolerance of 0.05 Da, peptide charges 2+, 3+, and 4+, on a QExactive instrument (Thermo, Waltham, MA, USA), and with decoy search to determine false discovery rate (FDR). For export, an ion-cutoff of 20 was chosen (exported peptide measurements: Data S9). The MS proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.prote omexchange.org) via the PRIDE partner repository [bib_ref] 2016 update of the PRIDE database and its related tools, Vizcaino [/bib_ref] with the dataset identifier PXD006859. Technical outliers were identified using correlation analysis and principal component analysis (PCA) of protein abundance data implemented by an R script (Data S5). The average Pearson correlation coefficient of each GI-TAP replicate with the other replicates of the same time point was above 95% for all GI-3F6H samples apart from sample 19E [fig_ref] Figure 2: GI-TAP time series [/fig_ref] , which was also clearly separated from all other samples by PCA and was therefore discarded. A custom R script performed further statistical analysis and plotting (Data S7). We used a t-test to determine for each protein, whether the maximum GI-TAP time point (omitting the 31-h time point) is significantly different from the WT control average using q-values [Benjamini-Hochberg (BH) corrected P-values]. 'Fold enrichment' is the ratio of the highest GI-TAP time point to the WT control average gives. To assess temporal changes, ANOVA was performed on arcsinh-transformed GI-TAP data, including the ZT 31 time point. To assess rhythmicity, we used the JTK_CYCLE tool [bib_ref] JTK_CYCLE: an efficient nonparametric algorithm for detecting rhythmic components in genome-scale data..., Hughes [/bib_ref] to analyze periods of 22-26 h (Data S8). The summary heatmap [fig_ref] Figure 2: GI-TAP time series [/fig_ref] used the heatmap.2 function of the pvclust v2.0 R package. Gene ontology (GO) analysis was performed using TOPGO (http://bioconductor.org/biocLite. R, version: 2.16.0, [bib_ref] Improved scoring of functional groups from gene expression data by decorrelating GO..., Rahnenf€ [/bib_ref] , using a node size of 3, as described by [bib_ref] Sample preparation for phosphoproteomic analysis of circadian time series in Arabidopsis thaliana, Krahmer [/bib_ref] (Data S6). Biological context was provided by subcellular locations annotated in the SUBA resource [bib_ref] SUBA4: the interactive data analysis centre for Arabidopsis subcellular protein locations, Hooper [/bib_ref] and interaction data in the BioGrid [bib_ref] The BioGRID interaction database: 2017 update, Chatr-Aryamontri [/bib_ref]. ## Yeast two-hybrid assay Full-length CDF6 coding sequence was PCR-amplified using cDNA as template with primers shown in , cloned into pENTR/D-TOPO (Invitrogen) and sequenceverified. The plasmid cassette was transferred to pAS-GW, a gateway compatible bait vector [bib_ref] Proteinprotein interactions between large proteins: two-hybrid screening using a functionally classified library..., Nakayama [/bib_ref] using LR clonase II (Invitrogen). The GI-FL, GI-N, GI-M + C, FKF1, LKP2, and ZTL clones used in this analysis were described previously; GI-FL, GI-N and GI-C [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] , and FKF1, LKP2, and ZTL [bib_ref] FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in..., Imaizumi [/bib_ref]. Yeast strains Y187 and AH109 were transformed with prey and bait vectors, respectively using the standard yeast transformation protocol (Clontech, Mountain View, CA, USA). After colonies formed on -W or -L containing media, three independent colonies were grown together, and then mated against their corresponding pairs for 3 days on YPDA media. After mating, yeast colonies were transferred onto -WL media. After checking for mating confirmation, yeast sectors were retransferred at the same time onto -WL and -WLH media. The experiments were repeated several times with the same results. # Modeling methods Simulations of the P2011 clock model [bib_ref] The clock gene circuit in Arabidopsis includes a repressilator with additional feedback..., Pokhilko [/bib_ref] for GI and GI-3F6H were performed in COPASI v4.16 [bib_ref] COPASI-a COmplex PAthway SImulator, Hoops [/bib_ref]. Simulations of the Framework Model FMv2for CO, FT, and flowering time were performed in MATLAB (Mathworks, Cambridge, UK). Both models are available online: P2011 (http://www.plasmo.ed.ac.uk/plasmo/models/download.sht ml?accession=PLM_71&version=1) and FMv2 (https://fa irdomhub.org/models/248?version=2). The higher arrhythmic GI RNA levels in GI-3F6H plants were simulated by reducing the affinity of GI for its rhythmic transcriptional inhibitors (parameters g14, g15) by 100-fold each, compared to the default, wild-type values. Transcriptional activation (parameter n12) was then reduced by 36% to match the observed GI-3F6H mRNA level. The effects on other model readouts (Figs 1 and 3) were caused by this simulated transcriptional mis-regulation of GI. Simulations of the flowering pathway were conducted using the photoperiod and temperature conditions of the corresponding experiments. # Results ## Characterization of the gi-3f6h transgenic plant line We transformed the strong gi-2 mutant (a deletion allele predicted to truncate~90% of GI protein) [bib_ref] GIGANTEA: a circadian clock-controlled gene that regulates photoperiodic flowering in Arabidopsis and..., Fowler [/bib_ref] with a construct to express 3xFlag-and 6xHis-tagged GI protein under the control of the CaMV 35S promoter (GI-3F6H). We aimed to express GI-3F6H protein constitutively at a similar level throughout the day to be able to immunoprecipitate a similar amount of GI at each time point [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref]. This will enable us to detect the changes in interaction of certain proteins with GI rather than the changes in the amount of coimmunoprecipitated proteins caused by the different amount of GI expressed. After isolating several positive transformants, we chose the line in which the expression levels of GI-3F6H transcripts were similar to the peak expression levels of the endogenous GI [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. As a first experiment, we performed a preliminary study where we used TAP of GI-3F6H followed by silver-staining of a protein gel separation and LC-MS of excised gel bands [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] , Data S1). Our GI-3F6H line expressed sufficient GI protein for effective TAP and analysis of gel bands by MS identified GI and known interactors [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] , Data S1). In addition, this GI-3F6H construct completely rescued the late flowering phenotype of gi-2, indicating that GI-3F6H is functional [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. In the GI-3F6H line, CO and FT mRNAs were higher than in the WT in the morning [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] , consistent with activation of these flowering-promoting genes by GI in the light [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] [bib_ref] GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana, Sawa [/bib_ref]. This was also reflected by slightly ELF of the GI-3F6H plants relative to the WT [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. GIGANTEA has multiple, known effects on the clock and flowering genes and proteins; several of these effects have been incorporated into mathematical models [bib_ref] The clock gene circuit in Arabidopsis includes a repressilator with additional feedback..., Pokhilko [/bib_ref]. In order to test whether the effects of the misregulated GI-3F6H transgene were replicated by these known mechanisms, we simulated the rescued mutant line in the Arabidopsis Framework Model version 2, a mechanistic, mathematical model that includes photoperiodic flowering [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. The WT and gi-2 simulations closely matched the mRNA data. This data set favored morning (1-4 h after dawn) expression of CO and FT compared to evening (13-16 h) expression slightly more than the model, possibly reflecting a reduced GI mRNA level at 13 h in this data set compared to previous training data. The model correctly predicted an elevated CO mRNA level at 1 h in GI-3F6H plants, though the observed level was~5-fold rather than~2-fold higher [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. This CO peak induced more FT at 4 h in the model [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] than in the plant [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] , and therefore earlier flowering [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. Morning regulation of FT, in the presence of unusually high GI levels, differed most between the model and the plant, highlighting this as an area for future model refinement. Next, GI-3F6H and WT plants (control) were grown in long days for 2 weeks and harvested at ZT 13 when GI protein peaks [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] and potential GI-interacting proteins were identified in a qualitative proteomics study [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. TAP of GI-3F6H was carried out, followed by on-bead digestion and qualitative MS analysis [fig_ref] Figure 2: GI-TAP time series [/fig_ref] , reporting peptide counts [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. Fifty Arabidopsis proteins were identified by at least one peptide in each of the GI-3F6H samples and none in the controls. In order to exclude known nonspecific interactors, we eliminated all proteins from our list that were previously reported to be purified by GFP-3F6H with the same sample preparation protocol [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] (Data S3). In addition, during the extraction, GI-3F6H can come in contact with proteins from compartments that are inaccessible to it in an intact cell. Therefore, we also excluded proteins from further analyses that are located in the chloroplast or mitochondria but not in the cytoplasm or nucleus according to their GO annotation [bib_ref] PANTHER: a library of protein families and subfamilies indexed by function, Thomas [/bib_ref] ; full results in Data S2). Eighteen proteins remained after this background removal strategy, indicating the potential to identify previously undiscovered interactions, as discussed below. Detection of known direct interactors, such as ZTL, FKF1, and LKP2, as well as indirect interactors, ASK1 and ASK2 (direct interactors of ZTL and FKF1) validated the methodology [bib_ref] Forward genetic analysis of the circadian clock separates the multiple functions of..., Kevei [/bib_ref] [bib_ref] Formation of an SCF ZTL complex is required for proper regulation of..., Han [/bib_ref] [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] [bib_ref] Expression and interaction analysis of Arabidopsis Skp1-related genes, Takahashi [/bib_ref]. ## Candidate gi-interacting proteins from time series data Although GI plays an important role in photoperiodic flowering in long days, the function of GI under shortday conditions remains elusive. Therefore, we grew plants in short days to identify uncharacterized interactors of GI potentially involved in other responses. In order to obtain time-resolved interaction data, we applied the same GI-TAP method as in the qualitative study to plants grown in short-day conditions, at six time points in biological quintuplicate, with additional duplicate samples at time point 31 h (replicating the 7-h time point; [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] 'time series', [fig_ref] Figure 2: GI-TAP time series [/fig_ref]. Short-day conditions ensure plants to be at a vegetative stage at the time of sampling, while being large enough to obtain sufficient amounts of tissue from a manageable number of plants for our time-resolved TAP-MS procedures. Extraction, TAP, and sample preparation for MS were carried out as for the qualitative analysis [fig_ref] Figure 2: GI-TAP time series [/fig_ref]. Using the Mascot search engine to identify peptides, our choice of peptide score cutoff of 20 resulted in an FDR of 0.023. After identification and quantification of proteins [fig_ref] Figure 2: GI-TAP time series [/fig_ref] , one outlier (GI-3F6H sample at ZT19, replicate E) was excluded from subsequent analysis (see Experimental Procedures; [fig_ref] Figure 2: GI-TAP time series [/fig_ref]. PCA of the remaining GI samples maximally separated the mid/late-night time points 19 h and 23 h from mid-day time points 7 h and its replicate 31 h [fig_ref] Figure 2: GI-TAP time series [/fig_ref]. Two thousand three hundred thirty-six peptides were detected in the time series study, from which 500 proteins were quantified . In order to exclude known unspecific interactors, we used the same strategy as for the qualitative study, eliminating 80 proteins previously purified by GFP-3F6H [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] and 169 chloroplast and mitochondrial proteins [bib_ref] PANTHER: a library of protein families and subfamilies indexed by function, Thomas [/bib_ref] (Data S3, S4 and S6). The analytical methods also quantified the identified peptide peaks in WT control samples (one replicate for each time point except ZT 15 and ZT 31) that had been subjected to the same TAP procedure (Figs 1F and 2A). Subsequent analysis used raw abundance data exported from Progenesis as opposed to the abundance which is normalized by the sum of all intensities of ions with the chosen inclusion criteria (see Materials and methods) in each mass spectrometric analysis; however, analysis of normalized data gave very similar results (data not shown and Data S3). The fold enrichment of each protein in the GI-3F6H was calculated as the peak GI-3F6H abundance relative to the average abundance in the WT control samples. Potential interacting proteins were identified as significantly enriched by t-test compared to the WT control, with a significance threshold adjusted for multiple testing (BH-adjusted q-value < 0.05). Fold-enrichment threshold values were informed by the results for known interactors. The direct interactors ZTL, FKF1, and LKP2 were more than 10-fold enriched over the WT control in the GI-3F6H time series. Indirect interactors CUL1/CUL2 and GLUTAMINE SYNTHETASE 2 (GLN2) [bib_ref] Formation of an SCF ZTL complex is required for proper regulation of..., Han [/bib_ref] [bib_ref] Distinct roles of FKF1, GIGANTEA, and ZEITLUPE proteins in the regulation of..., Song [/bib_ref] were two-to three-fold more abundant at their peaks than the time series control and were not identified in the preliminary or qualitative studies. Hereafter, we refer to significantly enriched proteins with at least four-fold enrichment as highly enriched and to proteins with two-to four-fold enrichment as weakly enriched (a further 88 proteins). Among these 88 proteins, 13 changed significantly in abundance within the time series (assessed for any change by ANOVA, q-value < 0.05) or 10 as assessed for rhythmic profiles (by JTK_CYCLE q-value < 0.05; and Data S3; Data S8). Peak abundance for most of the 16 proteins with changing or rhythmic precipitated protein abundance, as well as nonrhythmic/ changing precipitated proteins occurred at 7 h, with lowest average abundance at 23 h among the significantly enriched proteins, and no rhythmic or changing proteins peaking at 15 h [fig_ref] Figure 2: GI-TAP time series [/fig_ref]. The immunoprecipitated abundance of GI changed about two-fold over time [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. The observed abundance changes for immunoprecipitated GI and ZTL closely matched the predicted protein profiles [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref] from simulation of GI-3F6H (as in [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. This result indicated . Candidate interacting proteins identified in the qualitative study. Control (WT samples) and GI-3F6H samples were extracted in RIPA or SII buffer. Eighteen Arabidopsis proteins were identified by at least one peptide in each GI-TAP sample and none in the WT background controls, excluding proteins that are likely contaminants as they bind to GFP-3F6H [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] or localized to other compartments than GI (chloroplast, mitochondria). The right-hand columns cross-reference the time series study, with fold enrichment and significance (q-value) of the maximum GI-3F6H time point relative to the WT control. Bold: known direct or indirect interactors and homologs. n.d.: not detected. that the multiple mechanisms of GI and ZTL protein regulation in the model were sufficient to replicate the abnormal accumulation of GI-3F6H protein, and its effects on ZTL. The change in precipitated GI abundance was not significant by ANOVA or JTK_ CYCLE. ## Functional categorization of gi-tap enriched proteins Gene ontology analysis was done, using the candidates inas foreground. GO terms related to protein degradation were overrepresented among the candidates, as well as light response related terms, some metabolic processes, and flower development (Data S4 and S6). ## Rhythmic profiles of known interactors In contrast to the weakly rhythmic trend in abundance of the immunoprecipitated GI protein, known interactors showed contrasting profiles [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. The direct interactors FKF1, ZTL, and LKP2 showed temporal profiles consistent with their mRNA expression patterns [bib_ref] FKF1, a clock-controlled gene that regulates the transition to flowering in Arabidopsis, Nelson [/bib_ref] [bib_ref] ZEITLUPE encodes a novel clock-associated PAS protein from Arabidopsis, Somers [/bib_ref] [bib_ref] Linked circadian outputs control elongation growth and flowering in response to photoperiod..., Seaton [/bib_ref] [bib_ref] THE DIURNAL PROJECT: diurnal and circadian expression profiling, model-based pattern matching and..., Mockler [/bib_ref] [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. The ZTL profile paralleled GI, consistent with their mutual stabilization [bib_ref] The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA, Kim [/bib_ref] and closely matched by the prediction from the model simulation [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. Co-immunoprecipitated LKP2 abundance had a similar trend, consistent with arrhythmic mRNA expression of ZTL and LKP2. Only FKF1 and CDF3 were strongly rhythmic, with FKF1 peaking at 7-11 h, resembling previous data [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref] [bib_ref] FKF1 is essential for photoperiodic-specific light signalling in Arabidopsis, Imaizumi [/bib_ref] and CDF3 peaking at 27 h. Therefore, CDF3 level is in antiphase with FKF1, in line with the degradation of CDFs by FKF1 [bib_ref] FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in..., Imaizumi [/bib_ref] [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref]. These results demonstrate the consistency of our data with published results. CDF3 was quantified using a single, individually inspected peptide, indicating that such data should not be excluded from analysis. Several established indirect interactors of GI were quantified [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. CDF3 and GLN2 are client proteins of FKF1 [bib_ref] Distinct roles of FKF1, GIGANTEA, and ZEITLUPE proteins in the regulation of..., Song [/bib_ref] , with CDF3 also being a direct GI interactor (see above) [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] , whereas ZTL and LKP2 also interact with the core components of the SCF ubiquitin E3 ligase detected here, ASK1 and CUL1 and/or CUL2 (closely related proteins that were not distinguished by the peptides detected). ## The predicted functions of candidate interactors include protein degradation and stabilization In addition to verifying the known indirect interactor CUL1, our analysis enriched other proteins involved in protein stability [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. The ubiquitin-specific proteases (UBP) 12 and UBP13 (AT5G06600 and AT3G11910) were enriched in the time series and qualitative studies. Both UBP12 and UBP13 regulate the period length of the circadian clock as well as photoperiodic flowering, therefore the function of UBP12 and UBP13 in the clock and flowering regulation might be through the GI complex (see note added in proof). AAA-type ATPase family proteins related to components of the 26S proteasome (AT1G45000 and/ or AT4G27680) and a protease inhibitor, CYSTATIN 1 (AT5G12140) were also enriched. Several proteins annotated as being involved in protein stabilization were also significantly enriched by GI-3F6H, such as Cpn60 chaperonin family proteins (AT1G24510, AT3G18190, AT3G02530 and AT3G03960), the putative co-chaperone TPR4 (AT1G04530;, [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref] and at least one HSP90 (AT5G56030, AT5G52640 and/or AT5G56000, [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. HSP70 family proteins were just below the enrichment cutoff. In contrast, neither additional F-box proteins nor other proteins involved in circadian timekeeping were identified as strong candidate interactors (enrichment . Numbers of quantified and significant proteins in the GI-3F6H-MS time series. The raw data of the Progenesis output file and the raw data after removing likely unspecific proteins (GFP interacting proteins [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] and plastid or mitochondrial proteins, (see Data S3) was used for statistics. of PRR3 in the time series was below the significance threshold). Multiple, metabolic enzymes and translation elongation or initiation factors were enriched in the GI-3F6H time series. Among those, were GTP-binding translation factors (AT1G72730 and AT1G54270 and/or AT3G19760), TREHALOSE-6-PHOSPHATASE SYNTHASE 8 (TPS8; [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref] , a phosphofructokinase family protein (AT1G20950) and a GMP synthase homolog (AT1G63660,and a pyruvate kinase family protein (AT2G36580). In addition, a protein that binds to di-or trimethylated histone H3, ALFIN-LIKE 7 [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref]. This protein was the most significantly rhythmic of the candidate interactors after FKF1 and CDF3 (BH-adjusted P-value from JTK_CYCLE = 8 9 10 À6 ). Its immunoprecipitated protein levels were the most anticorrelated with FKF1 levels among the highly enriched proteins [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref] , followed by CDF3 (r = À0.65 and À0.40, respectively). The DOF protein AT1G26790 is a close homolog of CDF5 [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref] [bib_ref] CYCLING DOF FACTOR 1 represses transcription through the TOPLESS co-repressor to control..., Goralogia [/bib_ref] and its mRNA expression showed a robust circadian oscillation in constant light [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref]. Therefore, we named this gene CYCLING DOF FACTOR 6 (CDF6). We then validated the interaction of CDF6 with GI and ZTL/FKF1/LKP2 proteins as well as its function. Yeast-2-hybrid (Y2H) assay experiments confirmed the interaction of full-length, N-terminal, and C-terminal regions of GI with CDF6, as well as interaction of CDF6 with FKF1, ZTL, and LKP2 [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref]. CDF6 transcript abundance was tested in plants transferred to constant light, revealing circadian regulation with a sharp peak around subjective dawn [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref] , similar to CDF1 transcript abundance [bib_ref] FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in..., Imaizumi [/bib_ref] [bib_ref] Linked circadian outputs control elongation growth and flowering in response to photoperiod..., Seaton [/bib_ref] and the profile of CDF6 in the GI-3F6H time series [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref]. Since GI interacts with FKF1 and most likely CDF3 [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref] [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref] [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref] , and because the CDF6 amino acid sequence shows high similarity to other CDFs, we predicted that CDF6 also has a similar function to other CDFs. To assess our hypothesis, transgenic plant lines were generated, in which CDF6 was expressed from the SUCROSE-PROTON-SYMPORTER (SUC2) promoter that is active in phloem companion cells [bib_ref] FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering, Song [/bib_ref]. We chose the SUC2 promoter to drive CDF6, because other CDFs as well as likely target genes of CDF6 -CO and FTare specifically expressed in phloem companion cells [bib_ref] CONSTANS acts in the phloem to regulate a systemic signal that induces..., An [/bib_ref] [bib_ref] TERMINAL FLOWER2, an Arabidopsis homolog of HETEROCHROMATIN PROTEIN1, counteracts the activation of..., Takada [/bib_ref]. Two lines, SUC2:HA-CDF6 #8 and #11, accumulated higher levels of the CDF6 transcript at ZT4 and in later time points of a qPCR time series [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref] , fluctuating around 20-60% of the WT peak level, whereas CDF6 levels in WT were very low except [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] is shown, and the sum of peptide numbers detected in the qualitative study (Qual., [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] in GI-3F6H and WT (control). Selected proteins detected by single peptides are shown below, along with proteins suggested by other hypotheses (see Discussion) that were below thresholds in the time series (*) but were detected in the qualitative study. Likely unspecific interactors [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] and inaccessible proteins are left out in this at the ZT1 peak. Both transgenic lines flowered significantly later than WT under long photoperiods, with less effect under short photoperiods [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref]. If CDF6 acts in a similar way to CDF1 [bib_ref] FKF1 conveys timing information for CONSTANS stabilization in photoperiodic flowering, Song [/bib_ref] [bib_ref] CYCLING DOF FACTOR 1 represses transcription through the TOPLESS co-repressor to control..., Goralogia [/bib_ref] , we would expect it to inhibit transcription of both CO and FT. Indeed, CO mRNA levels were reduced at 10, 13 h and at night in the transgenic plants compared to WT, and FT expression was reduced more than 10-fold at 4 h and at later time points [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref]. These results are consistent with CDF6 participating in the photoperiodic regulation of flowering, where CDF6 protein levels in WT are regulated through interaction with GI and its interacting Fbox proteins. ## A gi, direct and indirect known gi interactors # Discussion Proteostasis, the set of protein-metabolic processes, is expected to be critical for diel rhythms in general, because the removal of transcriptional repressor proteins controls the slow timing of circadian feedback circuits [bib_ref] Dependence of the period on the rate of protein degradation in minimal..., Gerard [/bib_ref] [bib_ref] The relationship between FRQ-protein stability and temperature compensation in the Neurospora circadian..., Ruoff [/bib_ref]. GI indirectly mediates the degradation of transcriptional repressors through interacting with F-box proteins involved in protein ubiquitination: ZTL mediates targeted degradation of TOC1 [bib_ref] Targeted degradation of TOC1 by ZTL modulates circadian function in Arabidopsis thaliana, Mas [/bib_ref] and PRR5 [bib_ref] Targeted degradation of PSEUDO-RESPONSE REGULATOR5 by an SCF ZTL complex regulates clock..., Kiba [/bib_ref] with LKP2 and FKF1 contributing [bib_ref] F-box proteins FKF1 and LKP2 act in concert with ZEITLUPE to control..., Baudry [/bib_ref]. FKF1 targets CDF1 for degradation to regulate photoperiodic flowering, and this FKF1-dependent degradation requires functional GI [bib_ref] FKF1 and GIGANTEA complex formation is required for day-length measurement in Arabidopsis, Sawa [/bib_ref]. GI also has protein chaperone functions to stabilize ZTL and potentially other proteins [bib_ref] HSP90 functions in the circadian clock through stabilization of the client F-box..., Kim [/bib_ref] [bib_ref] GIGANTEA is a cochaperone which facilitates maturation of ZEITLUPE in the Arabidopsis..., Cha [/bib_ref]. Our studies identified further proteostatic proteins associated with GI, and suggested links to metabolic sensing, providing candidates for the unknown targets of GI's proteostatic functions [bib_ref] GIGANTEA is a cochaperone which facilitates maturation of ZEITLUPE in the Arabidopsis..., Cha [/bib_ref] and recalling previous data linking GI, metabolic inputs, and biological timing. Overexpression of tagged GI (GI-3F6H) under the 35S promoter in the gi-2 mutant background rescued the mRNA expression of CO and FT and flowering time phenotypes of the mutant. GI protein tended to greater abundance during the day than during the night, in line with its light-dependent stabilization by ZTL [bib_ref] The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA, Kim [/bib_ref] , with an evening peak time similar to native GI protein [bib_ref] Arabidopsis GIGANTEA protein is posttranscriptionally regulated by light and dark, David [/bib_ref]. The observed immunoprecipitated protein profile closely matched the prediction of a mechanistic clock model that was informed by diverse literature data [bib_ref] The clock gene circuit in Arabidopsis includes a repressilator with additional feedback..., Pokhilko [/bib_ref] , indicating that the GI-3F6H protein conformed to the dynamic, light-responsive behavior expected from previous results [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. ## Confirmation of known interactors and the new direct interactor cdf6 The detection of known, indirect interactors of GI such as CUL1/2 among the weakly enriched proteins but not in the qualitative studies validated the time series approach. Among the known, direct interactors of GI that were not detected in our studies, SVP, TEM1, and TEM2 [bib_ref] GIGANTEA directly activates Flowering Locus T in Arabidopsis thaliana, Sawa [/bib_ref] , COP1 and ELF3 [bib_ref] COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI..., Yu [/bib_ref] , ELF4 [bib_ref] ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration, Kim [/bib_ref] , CO [bib_ref] Distinct roles of FKF1, GIGANTEA, and ZEITLUPE proteins in the regulation of..., Song [/bib_ref] , and TCP4 [bib_ref] TCP4-dependent induction of CONSTANS transcription requires GIGANTEA in photoperiodic flowering in Arabidopsis, Kubota [/bib_ref] are observed or expected to be largely or exclusively nuclear, while SPY [bib_ref] SPINDLY and GIGANTEA interact and act in Arabidopsis thaliana pathways involved in..., Tseng [/bib_ref] and SOS2 [bib_ref] Release of SOS2 kinase from sequestration with GIGANTEA determines salt tolerance in..., Kim [/bib_ref] are partly nuclear-localized. Analysis of nuclear preparations may be necessary to enrich for these and other, nuclear interactors. Rapid, whole-cell extraction was employed here to facilitate handling the larger sample numbers required to conduct the time series study in quintuplicate [bib_ref] Improved scoring of functional groups from gene expression data by decorrelating GO..., Rahnenf€ [/bib_ref]. Transcriptional regulators were nevertheless detected, including the known interactor CDF3 [bib_ref] FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in..., Imaizumi [/bib_ref] and its homolog CDF6 (AT1G26790). Y2H assays validated the interaction of CDF6 with GI N-and C-terminal fragments, as well as with ZTL, FKF1, and LKP2 [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref]. Functional overlap with other CDFs was confirmed, as CDF6 overexpression in leaf phloem companion cells inhibited CO and FT transcription and delayed flowering in a photoperiod-dependent manner [fig_ref] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering [/fig_ref]. CDF6 transcript expression in long days and constant light peaks around dawn, similar to CDF1, CDF2, CDF3, and CDF5 [bib_ref] FKF1 F-box protein mediates cyclic degradation of a repressor of CONSTANS in..., Imaizumi [/bib_ref] [bib_ref] Arabidopsis DOF transcription factors act redundantly to reduce CONSTANS expression and are..., Fornara [/bib_ref] [bib_ref] Linked circadian outputs control elongation growth and flowering in response to photoperiod..., Seaton [/bib_ref]. CDF6 interaction with GI was in antiphase to FKF1 interaction, consistent with CDF6 being largely or specifically degraded via this F-box protein. Our qualitative study and others conducted when GI normally accumulates [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] coincide with peak FKF1 abundance, so would not have detected CDF6 or perhaps CDF3 (Figs 3A and 4A), confirming the utility of the time series approach. However, only 10 proteins (11%) were enriched with a rhythmic profile, so the strong rhythms of FKF1 and the CDFs were uncommon. Rhythmic transcription of GI might normally confer rhythmicity on other partner proteins as it does for ZTL [bib_ref] ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light, Kim [/bib_ref] [bib_ref] The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA, Kim [/bib_ref] , in which case we expect mis-expression of GI to alter partner protein accumulation, as GI-3F6H does to ZTL. Alternatively, many partner proteins might lack strong rhythmicity. The large size and proposed proteostasis functions of GI (discussed below) risk false-positive results. GI has not been found localized in or associated with the chloroplast but rather in the nucleus or cytoplasm [bib_ref] Distinct roles of GIGANTEA in promoting flowering and regulating circadian rhythms in..., Mizoguchi [/bib_ref] [bib_ref] The F-box protein ZEITLUPE controls stability and nucleocytoplasmic partitioning of GIGANTEA, Kim [/bib_ref] [bib_ref] GIGANTEA is a nuclear protein involved in phytochrome signaling in Arabidopsis, Huq [/bib_ref]. The abundant, plastid-localized proteins enriched as interactors (Data S3 and S4) likely reflect unspecific binding, at least in the case of chloroplastencoded proteins, which was an expected cost of detecting low-abundance and indirect interactors. Conservatively, we excluded mitochondrial and chloroplast proteins (see Materials and methods; Data S3) from the candidate interactors. GI might in principle have a physiological role in the metabolism of proteins translated on cytosolic ribosomes, prior to compartmentalization, or of proteins translocated from other compartments to the cytosol for degradation [bib_ref] Protein abundance changes and ubiquitylation targets identified after inhibition of the proteasome..., Svozil [/bib_ref]. ## Metabolic and nuclear functions of gi Functionally at least, GI links carbon metabolism and timing, via a long-term response of the circadian clock to sucrose [bib_ref] The circadian oscillator gene GIGANTEA mediates a long-term response of the Arabidopsis..., Dalchau [/bib_ref] and the photoperiodic adjustment of the rate of starch biosynthesis [bib_ref] Regulatory properties of ADP glucose pyrophosphorylase are required for adjustment of leaf..., Mugford [/bib_ref]. The trehalose-6phosphate pathway mediates several such sugar responses [bib_ref] Extensive expression regulation and lack of heterologous enzymatic activity of the Class..., Ramon [/bib_ref] [bib_ref] Regulation of flowering by trehalose-6-phosphate signaling in Arabidopsis thaliana, Wahl [/bib_ref]. TPS8 is a paralogue without known enzymatic activity but with diurnally regulated expression, repressed by sucrose [bib_ref] Global transcript levels respond to small changes of the carbon status during..., Usadel [/bib_ref]. GI interaction with TPS8 was highly enriched and, unusually, peaked at ZT19 [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref] , providing one of several possible mechanisms for GI to mediate between metabolism and biological timing. Few candidate interactors were shared with a previous study using ELF3 and ELF4 bait proteins [bib_ref] Identification of evening complex associated proteins in Arabidopsis by affinity purification and..., Huang [/bib_ref] , which each interact with GI [bib_ref] ELF4 regulates GIGANTEA chromatin access through subnuclear sequestration, Kim [/bib_ref] [bib_ref] COP1 and ELF3 control circadian function and photoperiodic flowering by regulating GI..., Yu [/bib_ref]. For example, RACK1A (AT1G18080) is a promiscuously interacting protein with several reported physiological roles in plants [bib_ref] The receptor for activated C kinase in plant signaling: tale of a..., Islas-Flores [/bib_ref]. Its homolog RACK1B (AT1G48630) was also weakly enriched. Mammalian RACK1 affects the circadian clock through the interacting core clock transcription factor BMAL1 [bib_ref] The RON1/ FRY1/SAL1 gene is required for leaf morphogenesis and venation patterning..., Robles [/bib_ref] , and contributes to degradation of its paralogue hypoxia- induced factor HIF1a. HIF1a protein regulation is mediated via HSP90 and UBP (reviewed in [bib_ref] Targeting protein-protein interactions in the HIF system, Wilkins [/bib_ref] : their Arabidopsis homologs were highly enriched in our GI-3F6H datasets. ## Potential function of gi in cold response Some of our candidate interactors may be used to speculate on new mechanisms contributing to GI function. GI enhances cold tolerance independently of CBF signaling [bib_ref] Involvement of GIGANTEA gene in the regulation of the cold stress response..., Cao [/bib_ref]. Two of our candidate interactors, REI1-LIKE and GENERAL CONTROL NON-REPRESSIBLE (GCN1;, have been implicated in cold tolerance through a role in ribosome maturation and regulation of translation initiation, respectively [bib_ref] The REIL1 and REIL2 proteins of Arabidopsis thaliana are required for leaf..., Schmidt [/bib_ref] [bib_ref] The inhibition of protein translation mediated by AtGCN1 is essential for cold..., Wang [/bib_ref]. Knowledge of these potential interactors may therefore be helpful to generate hypotheses on how GI mediates cold tolerance. ## Gi candidate interactors involved in protein metabolism Protein degradation of clock-relevant, transcriptional repressors was the first biochemical function supported for GI, acting as a scaffold for F-box proteins, though GI's co-chaperone function is now also implicated [bib_ref] ZEITLUPE is a circadian photoreceptor stabilized by GIGANTEA in blue light, Kim [/bib_ref] [bib_ref] GIGANTEA is a cochaperone which facilitates maturation of ZEITLUPE in the Arabidopsis..., Cha [/bib_ref]. No further F-box proteins or other ubiquitin E3 ligases were identified here, suggesting that GI mediates further physiological roles through different biochemical mechanisms. Chaperone proteins are typical, nonspecific contaminants of affinity purification studies but direct, physiologically relevant binding of HSP90 with GI has been demonstrated [bib_ref] GIGANTEA is a cochaperone which facilitates maturation of ZEITLUPE in the Arabidopsis..., Cha [/bib_ref]. HSP90 isoform(s) were highly enriched and weakly rhythmic in our time series [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. TPR4, which encodes a tetratricopeptide repeat (TPR) protein with potential to interact with HSP90/HSP70 as a co-chaperone [bib_ref] In silico identification of carboxylate clamp type tetratricopeptide repeat proteins in Arabidopsis..., Prasad [/bib_ref] was also strongly enriched, [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. The HSP70 family proteins that might function with GI and HSP90 [bib_ref] GIGANTEA is a cochaperone which facilitates maturation of ZEITLUPE in the Arabidopsis..., Cha [/bib_ref] were below the significance threshold in the time series studybut one (AT5G02500) was the fourth most enriched protein in the qualitative study . In contrast, several other proteins involved in proteostasis were highly and reproducibly enriched. For example, in our time series, two proteasome regulatory proteins were enriched, RPT1A (AT1G53750) and RPN10 (AT4G38630). GI-TAP had identified a different proteasome regulatory protein in rice [bib_ref] Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity..., Abe [/bib_ref]. TCP-1/cpn60 chaperonin family proteins (AT3G0 3960; AT3G20050) that can facilitate intercellular trafficking of transcription factors [bib_ref] Chaperonins facilitate KNOTTED1 cell-to-cell trafficking and stem cell function, Xu [/bib_ref] were detected in both the time series and the qualitative studies. Interestingly, a GI TAP-MS study in rice identified a potential GI interactor whose closest Arabidopsis homologs, ADL3 and ADL6, are also involved in post-Golgi vesicle trafficking [bib_ref] Identification of dynamin as an interactor of rice GIGANTEA by tandem affinity..., Abe [/bib_ref] [bib_ref] Arabidopsis dynamin-related proteins, DRP2A and DRP2B, function coordinately in post-Golgi trafficking, Huang [/bib_ref]. Our purification enriched several proteins involved in trans-Golgi or early endosome vesicle trafficking: RAB-A2B and/or RABA3 [bib_ref] Rab-A2 and Rab-A3 GTPases define a trans-Golgi endosomal membrane domain in Arabidopsis..., Chow [/bib_ref] , TUF [bib_ref] Arabidopsis vacuolar H+-ATPase subunit E isoform 1 is required for Golgi organization..., Strompen [/bib_ref] , and HAP13 [bib_ref] HAPLESS13, the Arabidopsis 1 Adaptin, is essential for protein sorting at the..., Wang [/bib_ref]. While we are not aware of any evidence for a Golgi/endosome related function of GI, these candidates may help to generate hypotheses on mechanisms of GI's to date unexplained functions. For example, Arabidopsis plants deficient in the Golgi-localized transporter protein PAR1 are more resistant to paraquat due to reduced plastid accumulation of the herbicide [bib_ref] PARAQUAT RESISTANT1, a golgi-localized putative transporter protein, is involved in intracellular transport..., Li [/bib_ref] , and a role of GI in stabilizing such intracellular transport proteins could be an explanation for the increased paraquat resistance of gi mutants in addition to the suggested increased resistance to oxidative stress [bib_ref] Oxidative stress tolerance and longevity in Arabidopsis: the late-flowering mutant gigantea is..., Kurepa [/bib_ref]. UBP12 and UBP13 were highly enriched in the time series and were also detected in the preliminary and/or qualitative studies (see note added in proof). Their deubiquitination activity potentially counteracts protein degradation, for example of Arabidopsis MYC2 [bib_ref] The deubiquitinating enzymes UBP12 and UBP13 positively regulate MYC2 levels in jasmonate..., Jeong [/bib_ref] , or monoubiquitination, for example of histone H2A. UBP12 and UBP13 are already known to affect the Arabidopsis circadian clock, act upstream of GI and CO in the same photoperiodic flowering time pathway [bib_ref] THE DIURNAL PROJECT: diurnal and circadian expression profiling, model-based pattern matching and..., Mockler [/bib_ref] , and are recruited to chromatin in association with the histone methylation complex PRC2. In a final connection, histone de-ubiquitination by USP7, the Drosophila homolog of UBP12/UBP13, is allosterically controlled by its interaction with a GMP synthetase [bib_ref] GMP synthetase stimulates histone H2B deubiquitylation by the epigenetic silencer USP7, Van Der Knaap [/bib_ref]. An Arabidopsis homolog (AT1G63660) of this enzyme was also enriched in the time series data. Our time series GI TAP-MS results not only identified a new member of CDF proteins functioning in the photoperiodic flowering pathway but also highlighted an extended set of proteostatic functions of GI, with intriguing potential links to metabolic enzymes that are now of interest in other organisms [bib_ref] Undercover: gene control by metabolites and metabolic enzymes, Van Der Knaap [/bib_ref]. These provide a novel set of hypotheses on the biochemical mechanisms of flowering regulation and of further physiological effects of GI. ## Note added in proof During preparation of this manuscript, UBP12 and UBP13 were independently identified as interactors of ZTL and LKP2 [bib_ref] Decoys Untangle Complicated Redundancy and Reveal Targets of Circadian Clock F-Box Proteins, Lee [/bib_ref] , suggesting that these UBP proteins are indirect interactors of GI. ## Data accessibility Research data pertaining to this article are located at figshare.com: https://dx.doi.org/10.6084/m9.figshare. 741597 ## Supporting information Additional supporting information may be found online in the Supporting Information section at the end of the article. [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref]. Validation of the GI-TAP procedure. [fig_ref] Figure 2: GI-TAP time series [/fig_ref]. Outlier analysis of the GI-TAP time series study. [fig_ref] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins [/fig_ref]. Transcript expression profiles of GI (A), FKF1 (B), ZTL (C), and FKF1 (D) from the diurnal website (http://diurnal.mocklerlab.org, [bib_ref] The deubiquitinating enzymes UBP12 and UBP13 positively regulate MYC2 levels in jasmonate..., Jeong [/bib_ref] , using the 'shortdays' condition. . Primer sequences. Data S1. List of proteins identified by LC-MS analysis of bands excised from silver-stained gel after GI-TAP (Preliminary study, [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] , includes original Mascot search output files. Data S2. List of proteins identified in the qualitative, on-bead digest analysis (Qualitative study, [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] , with peptide counts for GI-3F6H samples and WT background controls. Data S3. Proteins identified in the time series study [fig_ref] Figure 1: Evaluation of the GI-3F6H complementation line [/fig_ref] , with quantitation and statistics, put together from output generated by scripts in Data S7, and Data S8. Data S4. GO analysis on time series study: TopGO analysis results of GI-3F6H time series. Data S5. PCA on time series: R script, input files, output files; all on raw abundance data. Data S6. Gene ontology analysis on time series study, for Data S4. Data S7. Progenesis protein data export files, and analysis with R script for statistics on time series study (for Data S3 and. Data S8. JTK_CYCLE analysis of time series study. Data S9. Progenesis peptide measurements output file for time series study. [fig] 321 FEBS: Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] 322 FEBS: Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] 323 FEBS: Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] Figure 1: Evaluation of the GI-3F6H complementation line. (A,C,D) mRNA expression was tested in samples of Arabidopsis plants of the Col-0 WT (black open squares), gi-2 mutant (blue crosses) and gi-2 plants constitutively expressing the GI-3F6H fusion protein (red filled circles) under long-day conditions. qPCR assays detected GI (A), CO (C) or FT (D) mRNA. Data are means of biological triplicates, normalized to an IPP2 internal control; error bar, SEM. (B) Rosette leaf number was measured (data in bold) at flowering time in GI-3F6H, with WT and parental gi-2 mutant controls, under longday conditions. The flowering experiment was simulated using the FMv2 (pale colors). Data are averages of 16 plants; error bar, SEM. (E) GI transcription in the FMv2 was adjusted to match the GI mRNA profile of GI-3F6H in (A); predicted expression profiles of CO and FT are shown (as in C, D). (F) Overview of proteomics studies using GI-3F6H. 326 FEBS Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] Figure 2: GI-TAP time series. (A) Samples from GI-3F6H transgenic plants and WT control plants grown in short-day conditions were harvested at the indicated time points and replication. (B) Workflow for protein extraction, TAP using FLAG and His tags, and peptide preparation for MS. (C) Workflow for label-free, quantitative data analysis, statistical and bioinformatics tests. (D) Heat map of protein abundance over time for 88 proteins with significant enrichment of at least two-fold (excluding proteins that bind to GFP or are only found in inaccessible compartments). (E) Distribution of peak times, for 16 proteins shown in (D) with significant change over the GI-3F6H time course (ANOVA q < 0.05 or JTK_CYCLE q < 0.05). (F) PCA separates GI-TAP time points (grouped by number and contour, with replicate letter, all quantified proteins used) over components 1 and 2 (PC1, PC2). Note 31 h time point replicates 7 h. 327 FEBS Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] Figure 3: Diel profiles of immunoprecipitated GI and interacting proteins. Immunoprecipitated protein abundance of (A) GI and direct and indirect interactors detected in the time series study, along with (B) HSP90 and candidate direct or indirect interactors. Multiple gene identifiers indicate related proteins were not distinguished by the three CUL or five HSP90 peptides detected. GI-3F6H samples, markers; error bar, SEM. Average of WT control, horizontal line, AE SEM, dashed line. Time (T); Significance of enrichment and temporal change are shown, as q-values of t-test comparing GI-TAP peak to WT (Enrich q) and of JTK_CYCLE within the GI TAP-MS time series (Ch q). Biological replicates in (A and B):: 5, except for ZT 19 (four replicates) and ZT 31 (two replicates). (C) Simulation of the GI and ZTL protein time series, using the model matched to GI:3F6H RNA data (Fig. 1E) under short-day conditions, closely matches observations (panel A).328 FEBS Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] Figure 4: CDF6 interacts with GI and functions in photoperiodic flowering. (A) GI-interaction profile of CDF6 in the time series study is similar to CDF3 (Fig. 3A). n = 5 (except ZT19: n = 4) (B) Yeast two-hybrid assays validate interaction of CDF6 with full-length GI, N-and C-terminal domains of GI, as well as ZTL, FKF1 and LKP2. AD, activation domain; DBD, DNA binding domain. (C) Circadian expression profile of CDF6 mRNA, in WT plants 3 days after transfer to constant light, n = 3. (D) CDF6 over-expression delays flowering of transgenic SUC2:HA-CDF6 lines more under long days, compared to WT control, than under short days. Each transgenic line differed significantly from WT, t-test P < 0.0001, except #8 in SD, not significant. n ≥ 16 (E) mRNA expression profiles of CDF6, CO and FT were tested by qPCR in WT and the overexpressor lines, confirming that CDF6 suppresses evening CO and FT expression. n = 3. 331 FEBS Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] 332 FEBS: Letters 593 (2019) 319-338 ª 2018 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/fig] [fig] FEBS: Letters 593 (2019) 319-338 ª 2018 The Authors. Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. JK, AK, GSG, TI, AJM; Methodology, JK, YHS, RSJ, MJM, TLB; Software, JK, TLB; Formal Analysis, JK, GSG, AK, AZ, RSJ, AJM; Investigation, JK, GSG, AK; Data Curation, JK, TLB.; Writing, JK, GSG, AK, TI, AJM; Visualization, JK., GSG, AK, AJM; Supervision, KJH, TI, AJM; Funding Acquisition, JK, YHS, MJM, KJH, TLB, TI, AJM. [/fig] [table] Accession Name: Number of peptides identified in qualitative TAP experiment GI-3F6H time series enrichment (max. GI/WT) The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies. [/table] [table] Table 3: Known and candidate interactors of GI from GI-3F6H TAP-MS time course experiment. Quantified proteins with two or more peptides that were significantly enriched (in t-test of maximum GI-TAP time point with WT control q-value < 0.05) by at least two-fold (max. GI-TAP/WT > 2), ranked by fold enrichment. Only FKF1, CDF3 and CDF6 were rhythmic (JTK_CYCLE q-value < 0.05). Where peptides matched very similar proteins, multiple accession numbers are shown. Bold type, known direct or indirect interactors. Detection in the preliminary study (Prelim., [/table]

Effect of Vitamin D on the Vaginal Health of Menopausal Women: A Systematic Review Menopause is associated with the onset of climacteric symptoms due to low estradiol levels, which may cause insufficient maturation of the vaginal mucosa. Vitamin D may regulate the growth and differentiation of cells that are adversely affected due to low estradiol levels, thereby restoring vaginal health. The objective of this systematic review, the first on this subject, was to investigate the effect of vitamin D on the vaginal health of menopausal women. PubMed, Embase, Scopus, Web of Science, and Google Scholar databases and reference lists of hand-searched articles were searched for published studies from February 2000 to November 2018. The selection criteria were as follows: randomized or quasi-randomized trials that compared the effects of vitamin D or related compounds, alone or with calcium, on vaginal health (growth and differentiation of epithelial cells, dryness, acidity [pH]) outcomes in menopausal women. The methodological quality of these studies was examined using the Cochrane tool checklist by two independent investigators, following which the data were extracted. Of six examined studies, two showed that vitamin D administration improved the growth and differentiation of vaginal epithelial cells, improved vaginal pH, and decreased vaginal dryness in menopausal women. Although the level of evidence for the effects of vitamin D on vaginal health is low in our study, we concluded that vitamin D may improve the vaginal health of women, especially during menopause. # Introduction ## Description of the condition Menopause is an important period in the life of women; one reason being the numerous problems it may cause, affecting the quality of life of women. One common problem is vaginal discomfort, and it is estimated that up to 40% of post-menopausal women experience this problem, leading to loss of collagen, elastin, and smooth muscles in the vagina. Low estradiol levels, which menopausal women commonly experience, can cause a deficient maturation of vaginal mucosa. This condition is clinically presented by dryness, irritation, itch, and dyspareuniaand it could affect daily activities, sexuality, relationships, and quality of life. To evaluate vaginal health, it is necessary to assess vaginal epithelial cell measurement, vaginal dryness, and vaginal acidity (pH). ## Description of the intervention Treatment approaches that are used for vaginal health improvement in menopausal women are hormonal therapy and complementary therapy. Such treatments are commonly based on local hormonal therapy, in the form of vaginal creams, tablets, or suppositories, although other routes of hormone administration have also proven to be successful. Despite the fact that the benefits of estrogen replacement in preventing vaginal complications and reducing the incidence of related symptoms are well established Effect of Vitamin D on the Vaginal Health of Menopausal Women: A Systematic Review, hormonal therapy has various limitations and contraindications. Vitamins D and E have been used for the treatment of menopausal disorders and vaginal discomfort. Vitamin D is involved in the regulation of growth and differentiation of many cells, especially tissues lining the stratified squamous epithelium, which are present in the vagina and are regulated by Vitamin D. Vitamin D3 binds to an intracellular receptor that is a member of the steroid-thyroid hormone receptor family. Vitamin D3 and its receptors form a complex that binds to the Vitamin D3 response element of genes and either positively or negatively affects the transcription of that gene. The importance of this review: Being free of sexual problems can improve the confidence and mental state of women; therefore, it is important to resolve sexual problems associated with menopause. Accordingly, vitamin D may be useful in vaginal discomfort in menopause women; however, some experimental results are controversy. ## Objective Review of the literature showed that there was no study that summarized the effects of vitamin D on vaginal health in menopausal women; therefore, the aim of this systematic review study was to evaluate and summarize the effects of vitamin D on vaginal health in menopausal women. # Materials and methods In this narrative systematic review the following criteria were used to determine their eligibility into our study. Inclusion criteria: we performed a screening of titles or abstracts followed by a full-text review. Studies were considered eligible if they met the following criteria: they were clinical randomized, placebo-controlled, double-blind, single-blind, non-blinded trials, or cohort trials published in the English language biomedical journals until 2019; they were quasi-randomized trials, the study populations were menopausal women; vitamin D supplementation alone or plus calcium was studied; the study was published in English language journals without reference to a scientific quality index; and all clinically-used dosages and durations of vitamin D administration in menopausal women were considered. Exclusion criteria: vitamin D was used in combination with other drugs except calcium, data was insufficient, studies were conducted on animals or in in vitro models, and the diagnostic criteria were not applied to the study subjects. Case reports and articles on non-vaginal health were excluded. Participants are menopausal women. Types of intervention are use of vitamin D supplementation alone or plus calcium. Exposure is vaginal health such as vaginal epithelial cells, vaginal dryness and vaginal pH in menopausal women. Types of outcome measures are performance-based; outcome measure was the Vaginal Maturation Index and vaginal pH and vaginal dryness. Primary outcomes are change in vaginal epithelial cells in menopausal women. Secondary outcomes are change in vaginal pH and vaginal dryness. ## Search methods for identification of studies The electronic search engines included PubMed, Web of Science, Scopus, Google scholar, and Embase using the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guideline. Search strategy: the search strategy included a combination of key words, and medical subject headings: vitamin D, vaginal, and menopause. We checked all titles and abstracts. Full text copies were obtained when the studies were considered possibly relevant. Searching other resources: gray literature and the reference list of articles. ## Data collection and analysis Selection of studies Two review authors independently screened titles and abstracts for inclusion of all potential studies, and after they were identified they were then coded as 'retrieve' (eligible or potentially eligible/unclear) or 'do not retrieve' . Next, the full-text studies coded as 'retrieve' were obtained and the two review authors independently screened the full-texts and identified studies for inclusion, and identified and recorded reasons for exclusion of the ineligible studies. ## Data extraction and management The following data were extracted (if available): author, publication year, and methodology (including criteria for menopause diagnosis, sample size, study design, dosage of intervention, duration of intervention, and main outcomes). We resolved any disagreement through discussion or, if required, we consulted a third person and used the consensus strategy. We identified and excluded duplicates and collated multiple reports of the same study. We recorded the selection process in sufficient detail to complete a PRISMA flow diagram. ## Assessment of risk of bias in included studies One reviewer assessed the quality of the included studies according to criteria for selection, methods of outcome assessment, and data analysis, using the Cochrane scale. If there were disagreements between the two reviewers during the process of study selection, the issues were resolved through discussion among multiple investigators. ## Measures of treatment effect For vaginal health outcomes we used the epithelial cells score (%) or number of participants with vaginal health improvement after intervention or after comparison with the control group. ## Dealing with missing data To complete the information that was unavailable from the published reports, we contacted the researchers of the relevant studies and they provided the missing information. ## Assessment of heterogeneity Methodological diversity such as variability in the study design and risk of bias were observed during this review. Some variations in the materials and methods such as varied drug forms of vitamin D, different inclusion criteria, or measurement scale in some studies led to no meta-analysis of the review. # Results A total of 351 studies seemed to be potentially relevant in the first search. However, 340 studies were excluded (irrelevant subjects) after screening the titles and/or abstracts. Finally, full texts of the remaining 11 studies were evaluated in depth, 6 trials with a total of 391 participants met the inclusion criteria.shows the diagram of the retrieved articles included in the systematic review. ## Description of studies Results of the search The search was updated from February 2000 to November 2018. We screened a total of 351 records from the following databases: Web of Science (7), PubMed, EMBASE (7), Google Scholar (31), Scopus (289). Overall, this review has a total of six trials, 23 duplicated, 5 animal studies, 50 breast cancer studies that were excluded, 11 studies selected for full text assessment in which 6 studies were excluded. Seefor the study flow diagram. Six trials were included in this review: 2 studies were individually randomized controlled trials (RCTs)and 4 were quasirandomized. A total of 391 participants were part of the 6 trials. Overall, the included trials fall into two main groups: vitamin D alone and vitamin D plus calcium. Seefor excluded studiesand their reason. ## Risk of bias in included studies Allocation (selection bias)shows the results for the random sequence generation and allocation concealment for the Cochrane Risk of Bias tool. For random sequence generation, 6 trials were deemed to be at low risk and for allocation concealment and 6 trials were at unclear risk. ## Blinding (performance bias and detection bias) Four trials did not report any attempt to blind assessors to treatment assignment. In 2 trial reports, the participants or providers, or both, were blinded to treatment allocation. ## Incomplete outcome data (attrition bias) Two trials did not provide information about the number of participants allocated to groups via randomizationand 4 trials reported attrition bias. ## The potential sources of bias In 6 studies, the inclusion and exclusion criteria and selective outcomes were clearly defined. The intervention and control groups were demonstrably comparable in 2 trials. ## Effects of interventions vitamin d alone versus placebo or no treatment Two studies used vitamin D alone vs. a placebo or no treatment. In the study of Rad et al., 1,000 IU doses of vitamin D vaginal suppository were used with for eight weeks, but in Bala et al.'s study 60,000 IU doses of vitamin D were administered orally and weekly. In two studies that included 244 menopausal women, both studies showed vitamin D was effective in the vaginal health of these women. ## Vitamin d plus calcium versus baselin Four studies used vitamin D plus calcium and compared the results versus baseline. In the study of Checa et al., 40 menopausal women used 500 mg of elemental calcium plus 400 IU vitamin D3 for 24 weeks. In Mucci et al.' s method, 141 mg calcium plus 400 IU of vitamin D were used daily, for 24 weeks in 45 women. In Carranza-Lira et al.' s study, 12 women used 1000 IU of calcitriol plus 500 mg of calcium daily for eight weeks, and in the study of Saeideh et al., 50 cases used 500 mg of calcium plus 200 IU of vitamin D daily, for 24 weeks. The 4 studies included 147 women. One study showed vitamin D plus calcium effectiveness in vaginal healthand 3 showed no positive result regarding vaginal health. ## Epithelial cell Rad et al.showed that use of vitamin D can improve the superficial, intermediate, parabasal of vaginal cells. Also, the mean vaginal maturation score between the case and control group was significantly increased. Bala et al.reported that use of vitamin D significantly affected the vaginal health index such as vaginal epithelial integrity. Checa et al.reported vaginal maturation mean score after 24 weeks (-38.7%; after 12 months, -51.8% significantly decreased). Saeideh et al.showed vaginal maturation mean score was not significant between the before intervention (27.51 ± 18.98) vs. after intervention . In Carranza-Lira et al.' s study, the mean % score change maturation index and parabasal and intermediate cells were not significant but the mean % score in superficial cells before intervention 2.5 ± 4.5% vs. after intervention 10.0 ± 8.5% was significantly changed. Three studies reported vitamin D can positively affect vaginal epithelial especially in superficial cells, although two other studies were inconsistent regarding this result. ## Vaginal ph Vaginal acidity (pH) was affected in two studies and pH mean score was significantly decreased in the vitamin D group. In the study of Carranza-Lira et al., the mean score of the pH strip test (1.5 ± 1.2 vs. 1.7 ± 1.0) did not significantly change before and after the intervention. Two studies reported significant change in vaginal pHand one study result was negative. ## Vaginal dryness Bala et al.found that use of vitamin D orally significantly affected vaginal moisture and consistency. Checa et al.showed that after 24 weeks, 21.4% of cases and after 12 months, 15.8% of cases suffered from vaginal dryness, but this change was not significant (P = 0.5). In Mucci et al.'s study, 13.3% of cases reported improvement after intervention vs. baseline and 39% of the vaginal dryness mean score decreased and this change was significant (P = 0.01). Saeideh et al.reported the number of vaginal dryness before intervention was 36 cases and after intervention was 37 cases, and Carranza-Lira et al.reported severity mean score using the visual analogue scale before intervention (58.5 ± 24.0) vs. after intervention (64.4 ± 27.2) was not significant. Two studies showed vitamin D can improve vaginal drynessand three studies were inconsistent. # Discussion ## Summary of main results Vitamin D alone versus placebo or no treatment Vitamin D alone, in the high doses tested or vaginaluse format, appears to be have an effect on vaginal epithelial cells especially superficial cells and vaginal pH decrease (two trials, 244 participants). Use of vitamin D alone in the vaginal formats and 1,000 IU doses tested daily appears not to be able to reduce vaginal dryness (one study, 44 participants). Duration time of use of vitamin D was 8-10 weeks, which seems sufficient time for vaginal health improvement except for vaginal dryness. ## Vitamin d plus calcium versus baseline Although there was no firm evidence that vitamin D (including 25-hydroxy vitamin D) with calcium was more effective than vitamin D alone for vaginal health in menopausal women, only one study (calcium plus vitamin D) reported positive vaginal health resultswhile three studies did not report any positive results. The calcium dose in all of the studies was 500 mg daily except for one studythat used a low dose (141 mg daily). The dose of vitamin D in two studieswas the same (400 IU), but their results were different. The study with low dose calcium and 400 IU vitamin D was effective in reducing vaginal dryness. Intervention durations were different, such as 8 weeks, 24 weeksand the longest being 48 weeks. Only 24 weeks duration of vitamin D plus calcium had positive results in vaginal dryness. ## Adverse effects Three trials reported adverse effects, which was mostly gastric discomfort. Overall, none of the trials reported the effect of vitamin D on mortality; however, use of calcium and vitamin D for a long time is dangerous and is not recommended for menopausal women, but in another study, which was not included in our study, the use of vitamin D and calcium was associated with a reduction in mortality (risk ratios 0.94, 95% CI 0.89 to 0.99). It is highly recommended to measure the level of vitamin D before high doses are administered and it is better to prescribe the supplement based on blood vitamin D levels. Calcium supplements could increase the risk of myocardial infarction, particularly in people with a higher dietary calcium intake. Supplements of calcium and vitamin D could also increase the risk of myocardial infarction and strokebut might also decrease the risk of breast cancer. ## Overall completeness and applicability of evidence This review included an evidence base from six trials that examined vitamin D (including 25-hydroxy vitamin D) with or without calcium in the improvement of vaginal health in menopausal women with a mean age of 50 years or over. Vitamin D was administered in a variety of formats and doses (oral daily, oral weekly, and vaginal suppository). However, three of the trials in this review tested daily vitamin D3 in doses greater than 800 IUand one of these trials recruit participants with very low (mean < 12 ng/mL) baseline 25-hydroxy vitamin D blood levels. American Geriatric Society suggests that a minimum blood vitamin D level of 30 ng/mL (75 nmol/L) is necessary in older adults to minimize the risk of menopause complications. It has been suggested that higher doses of vitamin D are required in women with vitamin D insufficiently, and vitamin D level testing before intervention is necessary for future studies in vaginal health in menopausal women. Mean duration of 24 weeks for oral use with a low dose of calciumand high dose vitamin D aloneis optimal for vaginal health in menopause women. ## Quality of the evidence This review included a small body of evidence from six trials with 391 participants. Our assessments of risk of bias and quality assessment are presented in. ## Potential biases in the review process We believe that selection bias is very low in this review. We have searched a wide range of databases for relevant journals. One report of studies that was published at a different time but using the same method and result was excluded. We only included RCTs and the other low quality evidences such as observational or case-control were excluded. ## Agreements and disagreements with other studies or reviews Although the results of two included studiesand two excluded studiesshowed improved superficial vaginal cells by using vitamin D, another study reported that vitamin D was not associated with menopausal related symptoms in menopausal women. The Cochrane review reported that supplements of vitamin D and calcium may prevent hip or other types of fractureand another study suggested that high intakes of dietary vitamin D and calcium may be modestly associated with a lower risk of early menopause. However, the use of complementary therapy such herbal medicine and supplements is beneficial for menopause symptoms such as hot flashes, but the results of a meta-analysis study conducted in 2017 showed that oral phytoestrogen cannot be effective in severe vaginal atrophy. Other treatments for vaginal atrophy in postmenopausal women may be the use of laser therapy, in which a study published in 2017, found the effectiveness of this method in improving vaginal health in postmenopausal women and improving their sexual quality, but the use of this method is not recommended due to need of advanced equipment that comes with a high cost. # Limitations One of the limitations of this systematic review was using varied drug forms of vitamin D in the evaluated studies. Other limitations were the different ages of the menopausal women and different inclusion criteria in the studies. The weak methodology of some studies used in our systematic review is also a limitation of this study. Small sample sizes, inadequate treatment allocation, unclear blinding method, and unmentioned randomization technique can degrade the validity of the results. Since there was large variation on the methodology of examined studies, it was impossible to do a meta-analysis. ## Implications for practice Vitamin D alone in the high doses and combined formulations that have been used in some studies appears to be effective in vaginal health in menopausal women. ## Implications for research There is a need to confirm vitamin D treatment effects on vaginal health using longitudinal studies. Result of this study show that the superficial epithelial cell can be improved with vitamin D. It has been suggested that the topical use of vitamin D in future studies be also compared with vaginal estrogen and vitamin D suppository.

Doctor and practice characteristics associated with differences in patient evaluations of general practice Background: Variation in patients' evaluation due to general practitioner (GP) and practice factors may provide information useful in a quality improvement context. However, the extent to which differences in patients' evaluation of the GPs are associated with differences in GP and practice characteristics must also be ascertained in order to facilitate comparison of adjusted patient evaluations between GPs. The aim of this study was to determine such associations in a setting where GPs serve a list of patients and act as gatekeepers.Methods:We carried out a patient evaluation survey among voluntarily participating GPs using the EUROPEP questionnaire, which produced 28,260 patient evaluations (response rate 77.3%) of 365 GPs. In our analyses we compared the prevalence of positive evaluations in groups of GPs.Results: Our principal finding was a negative association between the GP's age and the evaluation of all aspects, except accessibility. We also found an association between the way the practice was organised and the patients' evaluation of accessibility, with GPs in single-handed practices getting far the most positive evaluations. Long weekly working hours were associated with more positive evaluations of all dimensions except accessibility, whereas more than 0.5 full-time employees per GP, a higher number of listed patients per GP and working in a training practice were associated with negative evaluation of accessibility.Conclusion: GP characteristics are mainly associated with patients' experience of interpersonal aspects of care, while practice characteristics are associated with evaluation of accessibility. These differences need to be accounted for when comparing patient evaluations of different practices. # Background Ongoing efforts to improve the quality of general practice care increasingly include patients' evaluation of various aspects of care . Variation in patient evaluation of general practice reflects differences in performance, which, to some extent, may be associated with GP and practice characteristics, and differences in the patients' perception of performance. Crude unadjusted results, though, may serve best for quality improvement at a practice level as they reflect the extent to which the GPs succeed in meeting the patients' individual needs and dealing with different working conditions [bib_ref] Adjustment for patient characteristics in satisfaction surveys, Perneger [/bib_ref]. When comparing practices and when holding practices against standards comparability is obtained through standardisation -that is adjustment for systematic variation in patient, GP and practice characteristics between the compared groups. Studies of associations between patient evaluations and patient, GP and practice characteristics are needed to determine which characteristics to adjust for. Knowledge of such associations may also serve quality improvement purposes at regional and national levels. Earlier studies report a positive association between patients' evaluation and the GP's age [bib_ref] What type of general practice do patients prefer? Exploration of practice characteristics..., Baker [/bib_ref] , but a negative association between the evaluation and the GP's age and seniority has also been found [bib_ref] Characteristics of practices, general practitioners and patients related to levels of patients'..., Baker [/bib_ref] [bib_ref] Practice patterns, physicians' characteristics and patient-evaluated quality of general practice in Norway, Kvamme [/bib_ref]. Baker [bib_ref] What type of general practice do patients prefer? Exploration of practice characteristics..., Baker [/bib_ref] [bib_ref] Characteristics of practices, general practitioners and patients related to levels of patients'..., Baker [/bib_ref] and Campbell [bib_ref] Practice size: impact on consultation length, workload, and patient assessment of care, Campbell [/bib_ref] found an inverse relation between assessment and total list size as well as list size per GP. Lin [bib_ref] Patient perceptions of service quality in group versus solo practice clinics, Lin [/bib_ref] found better evaluations of group practices than of singlehanded practices, while Hombergh [bib_ref] Saying 'goodbye' to single-handed practices; what do patients and staff lose or..., Van Den Hombergh [/bib_ref] found the opposite, which was also supported by Wensing, who found better ratings of practices with fewer and full-time working GPs. This supports the general finding that patients prefer personal, continuous care [bib_ref] Saying 'goodbye' to single-handed practices; what do patients and staff lose or..., Van Den Hombergh [/bib_ref] [bib_ref] Continuity of care in general practice: effect on patient satisfaction, Hjortdahl [/bib_ref]. In addition, Baker [bib_ref] What type of general practice do patients prefer? Exploration of practice characteristics..., Baker [/bib_ref] [bib_ref] Characteristics of practices, general practitioners and patients related to levels of patients'..., Baker [/bib_ref] found that being a training practice produced less positive evaluations. These results derive from studies in different settings and with different degrees of adjustment for differences in patient characteristics. Hence, there seems to be a need for a sufficiently powered comprehensive study of associations between patient evaluations and GP and practice characteristics adjusted for systematic variation in patient characteristics. Such a study will provide us with information about GP and practice factors of significance to the quality of general practice and information on how to standardize patient evaluation results for comparison between (groups of) practices. On this background the aim of the present study is to determine to which extent variation in patient evaluations of the GPs are associated with GP and practice characteristics. # Methods ## Study population This study was carried out in a general practice setting where self-employed GPs work as gatekeepers for the public health services on a contract basis serving patients on their list (a brief introduction to the Danish general practice is given in Additional file 1). During 2002-4 all 2181 GPs from ten Danish counties received an invitation to carry out patient evaluations of their practices. A total of 365 GPs (16%-34% of all GPs in these counties) entered the project and undertook an evaluation. This was part of the national project on patient evaluations, the DanPEP. The participating GPs handed out questionnaires to 100 successive patients seen by the GP in the surgery or at home visits. The patients were at least 18 years of age, were listed in the practice and accepted a Danish language questionnaire. They were informed that their replies were anonymous to the doctor. Each questionnaire was identified by a serial number connecting it with the GP who handed it out and with the patient. All questionnaires not distributed within two weeks could be returned to the research office. All GPs filled in a form with information about the GP and the practice. Practice information from GPs working in the same practice was cross-checked. ## The questionnaire The questionnaire contained the 23 items forming the EUROPEP instrument, which is a European validated instrument for patient evaluation of general practice care based on literature analysis and surveys of patients' expectations and opinions on good care. The 23 items are displayed in Additional file 2. These questions covered specific aspects of general practice and were grouped into five dimensions: doctorpatient relationship, medical care, information and support, organisation of care and accessibility. The answers were marked on a 5-point scale ranging from "poor" to "excellent", with "acceptable" as the middle value. Alternatively, the patients could choose a sixth category "not able to answer/not relevant". The questionnaire also included questions about the patient's gender, age, educational level, frequency of attendance to a general practice, time listed with the GP, self-rated healthand chronic conditions. The patients were asked to assess the GP they considered to be their personal GP (and his practice when assessing aspects of accessibility) based on their contact experience over the past 12 months. They were also asked to write the GP's name on the questionnaire to confirm which GP was assessed and to allow individual assessment of GPs in partnership practices. The questionnaires were returned by the patients in prepaid envelopes to the research office. In order to be able to carry out the reminder procedure, the GPs recorded the names, addresses and serial numbers from the questionnaires handed out. Reminders with new questionnaires were sent out by the research office to nonresponding patients three to five weeks after the GPs' distributed the questionnaires and the patient lists were then destroyed. At the research office, the diagnoses reported by patients with chronic conditions were coded according to the major ICPC-2 groups. ## Assessments For each dimension, a patient's evaluation was included in the calculations only if 50% or more of the items had been answered in one of the six categories. An answer was considered positive if it fell into one of the two most favourable categories. An assessment of a dimension was categorised as 100%, 50-99% or 0-49% positive depending on the proportion of positively evaluated items in that dimension. In the analyses we compared the prevalence of assessments in the 100% category between strata and the prevalence in the 0-49% category, respectively. ## General practitioners The gender and age of the GPs, their seniority in present practice, number of weekly working hours (out-of-hoursduties, teaching away from practice and consultative services were not included), number of listed patients and full-time working staff per GP were categorised as displayed in [fig_ref] Table 1: Distribution of characteristics among participating general practitioners [/fig_ref] (in shared single-handed practices we only counted one GP). Practices were registered as urban, rural or mixed. They were divided into categories of single-handed, shared single-handed, groups of singlehanded or partnership practices with doctors working either full-time or part-time (practice types are explained in Additional file 1). Involvement in education was registered as education given in the practice and/or in settings outside the practice or as no involvement. ## Analyses Associations between the GP and practice characteristics and the assessment scores for each of the five dimensions were estimated as prevalence ratios (PR). The PRs with 95% confidence intervals (95% CI) were chosen instead of odds ratios, which would tend to overestimate the associations owing to the high prevalence of the variables. In the first model we estimated the crude PRs between the GP and practice characteristics and the proportion of 100% and 0-49% positive evaluations of each dimension. Based on these univariate analyses we identified confounding GP and practice variables. Hence, we found statistically significant correlations between GP age, gender and seniority and between organisation of practice, list size per GP, weekly working hours, urbanity and staff per GP and a high collinearity between GPs' age and seniority. GP age was more closely associated with patient evaluation than seniority and was therefore chosen as the adjusting variable. List size and urbanity were not associated with assessment and, hence, despite correlation with other variables, did not act as confounders. In the next model we estimated the association between the GP and practice characteristics and the evaluation adjusted for patient characteristics (patient-adjusted PR). The confounding patient variables (patients' age and gender, frequency of attending a GP and self-rated health) were identified from analyses of associations between patient characteristics and their GP evaluation performed on the data set also used in the present study . In the last model we included the confounding GP and practice variables resulting from the analyses in the first model, which were GP gender, age and weekly working hours, organisation of practice and staff (number of employees converted into full-time) per GP, along with the patient variables used in the former model and calculated the fully adjusted PRs for the associations between GP and practice characteristics and the patients' evaluations. ## Dependent variable We did two sets of analyses. In the first set the dependent variable was the prevalence of assessments in the 100% category. In the second set it was the prevalence of assessments in the 0-49% category. ## Independent variables Depending of the model (crude, patient adjusted or fully adjusted), our independent variables were GP gender, age and weekly working hours, organisation of practice and staff per GP and patient age, gender, frequency of attending a GP and self-rated health. ## Modelling data We used generalised linear models (GLM) with log link for Bernoulli family, i.e. modelling the PRs. Due to the high prevalences, some of the adjusted GLM analyses could not converge using the Bernoulli family. In these situations, we used the Poisson regression [bib_ref] Alternatives for logistic regression in cross-sectional studies: an empirical comparison of models..., Barros [/bib_ref] [bib_ref] A modified poisson regression approach to prospective studies with binary data, Zou [/bib_ref]. Model fit for each multivariate model was tested using the Hosmer-Lemeshow test for goodness of fitdeveloped for testing logistic modelling. However, where the Poisson regression was used, the high prevalences could produce estimated probabilities greater than one which would hamper the Hosmer-Lemeshow goodness of fit test very much. In such situations, we therefore used the post-estimation goodness of fit test for Poisson regression based on deviance statistics. Patient clustering by GPs produced relatively high intraclass correlation coefficients (ranging from 0.034 to 0.134). We accounted for patient clustering by using robust standard errors in all analyses. Analyses were performed using complete data only, i.e., the univariate and the GLM analyses were performed using the same set of data so that an increasing number of missings would not explain differences in associations when adjusting for more variables. We used Stata 9.1 for data processing. # Results A total of 36,561 questionnaires were distributed by the 365 GPs. After a reminder procedure and exclusion of responses from patients not indicating which GP they assessed or assessing non-participating GPs, we had 28,260 (77.3%) valid responses. Characteristics of the evaluated GPs and their practices are displayed in [fig_ref] Table 1: Distribution of characteristics among participating general practitioners [/fig_ref]. Respondent characteristics are shown in [fig_ref] Table 2: Distribution of characteristics among participating patients [/fig_ref]. There were more than twice as many female as male respond- [fig_ref] Table 2: Distribution of characteristics among participating patients [/fig_ref] , which reflects that women are more inclined than men to attend a GPand to respond to questionnaires (Heje et al., submitted). GP gender was not associated with evaluation outcome when we adjusted for patient, GP and practice confounders. However, adjustment did not neutralize the negative association found with GP age for all dimensions except accessibility. GPs aged 30-39 years received the most positive evaluations, whereas there was no difference between the other age categories. There was no association with seniority, neither before nor after adjustment. In the following the word "association" refers to the fully adjusted PR, i.e. the PR adjusted for confounding patient, GP and practice variables and for patent clustering. For the first four dimensions ("GP-patient-relationship", "medical care", "information and support" and "organisation of care"), we found a positive association between the evaluation and GP working hours in excess of 45 hours a week [fig_ref] Table 3: Dimension 1 [/fig_ref] , 5, 6). Patients' evaluation of the organisation of practice (e.g. continuity) was positively associ-ated with the GP's weekly working hours exceeding 37 hours. There was no association between weekly working hours and patients' assessment of accessibility [fig_ref] Table 7: Dimension 5 [/fig_ref]. We saw practically no association with practice urbanisation. The number of patients and the number of employees per GP were negatively associated with the experienced accessibility [fig_ref] Table 7: Dimension 5 [/fig_ref] , but not with the evaluation of the other dimensions [fig_ref] Table 3: Dimension 1 [/fig_ref] , 5, 6). The way the GPs had arranged themselves in practice played a minor role regarding the first four dimensions [fig_ref] Table 3: Dimension 1 [/fig_ref] , 5, 6), while there were major differences regarding accessibility. GPs in single-handed practices were assessed more positively than GPs in shared single-handed, groups of single-handed and partnership practices. Accessibility to GPs working part-time in partnership practices was assessed least favourably [fig_ref] Table 7: Dimension 5 [/fig_ref]. Working in a training practice was only associated with the assessment of accessibility aspects [fig_ref] Table 7: Dimension 5 [/fig_ref] , where we found a negative association, while the evaluations were not associated with teaching outside the practice. Prevalence ratio (PR) for 100% positive assessments and for 0-49% positive assessments associated with different patient characteristics. Crude PRs are adjusted for patient clustering. Patient-adjusted PRs are adjusted for patients' gender, age, frequency of attending a general practitioner (GP) and self-rated health and patient clustering. Fully adjusted PRs are adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. The goodness of model fitting is shown regarding the fully adjusted PRs (p-values). * Patients who marked 100% of the answered questions in one of the two most positive answering categories. † Patients who marked less than 50% (0-49%) of the answered questions in one of the two most positive answering categories. ‡ Prevalence § Crude prevalence ratio is the unadjusted prevalence ratio adjusted for patient clustering || Patient-adjusted prevalence ratio is the prevalence ratio adjusted for patients' gender, age, frequency of attending a GP and self-rated health and patient clustering. ¶ Fully adjusted prevalence ratio is the prevalence ratio adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. ** 95% confidence interval for the fully adjusted prevalence ratio. † † Poisson regression with robust variance ‡ ‡ Seniority in present practice § § "Single" = single-haned, "Shared single" = two ore more GPs sharing a single-handed practice, "Group of singles" = single-handed GPs sharing premises and staff, "Partnership" = GPs sharing patient-list, premises and staff, "Part-time partnership" = partnership practices with all GPs working part-time |||| Number converted to full-time working employees Prevalence ratio (PR) for 100% positive assessments and for 0-49% positive assessments associated with different patient characteristics. Crude PRs are adjusted for patient clustering. Patient-adjusted PRs are adjusted for patients' gender, age, frequency of attending a general practitioner (GP) and self-rated health and patient clustering. Fully adjusted PRs are adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. The goodness of model fitting is shown regarding the fully adjusted PRs (p-values). * Patients who marked 100% of the answered questions in one of the two most positive answering categories. † Patients who marked less than 50% (0-49%) of the answered questions in one of the two most positive answering categories. ‡ Prevalence § Crude prevalence ratio is the unadjusted prevalence ratio adjusted for patient clustering || Patient-adjusted prevalence ratio is the prevalence ratio adjusted for patients' gender, age, frequency of attending a GP and self-rated health and patient clustering. ¶ Fully adjusted prevalence ratio is the prevalence ratio adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. ** 95% confidence interval for the fully adjusted prevalence ratio. † † Poisson regression with robust variance ‡ ‡ Seniority in present practice § § "Single" = single-haned, "Shared single" = two ore more GPs sharing a single-handed practice, "Group of singles" = single-handed GPs sharing premises and staff, "Partnership" = GPs sharing patient-list, premises and staff, "Part-time partnership" = partnership practices with all GPs working part-time |||| Number converted to full-time working employees Prevalence ratio (PR) for 100% positive assessments and for 0-49% positive assessments associated with different patient characteristics. Crude PRs are adjusted for patient clustering. Patient-adjusted PRs are adjusted for patients' gender, age, frequency of attending a general practitioner (GP) and self-rated health and patient clustering. Fully adjusted PRs are adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. The goodness of model fitting is shown regarding the fully adjusted PRs (p-values). * Patients who marked 100% of the answered questions in one of the two most positive answering categories. † Patients who marked less than 50% (0-49%) of the answered questions in one of the two most positive answering categories. ‡ Prevalence § Crude prevalence ratio is the unadjusted prevalence ratio adjusted for patient clustering || Patient-adjusted prevalence ratio is the prevalence ratio adjusted for patients' gender, age, frequency of attending a GP and self-rated health and patient clustering. ¶ Fully adjusted prevalence ratio is the prevalence ratio adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. ** 95% confidence interval for the fully adjusted prevalence ratio. † † Poisson regression with robust variance ‡ ‡ Seniority in present practice § § "Single" = single-haned, "Shared single" = two ore more GPs sharing a single-handed practice, "Group of singles" = single-handed GPs sharing premises and staff, "Partnership" = GPs sharing patient-list, premises and staff, "Part-time partnership" = partnership practices with all GPs working part-time |||| Number converted to full-time working employees Prevalence ratio (PR) for 100% positive assessments and for 0-49% positive assessments associated with different patient characteristics. Crude PRs are adjusted for patient clustering. Patient-adjusted PRs are adjusted for patients' gender, age, frequency of attending a general practitioner (GP) and self-rated health and patient clustering. Fully adjusted PRs are adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. The goodness of model fitting is shown regarding the fully adjusted PRs (p-values). * Patients who marked 100% of the answered questions in one of the two most positive answering categories. † Patients who marked less than 50% (0-49%) of the answered questions in one of the two most positive answering categories. ‡ Prevalence § Crude prevalence ratio is the unadjusted prevalence ratio adjusted for patient clustering || Patient-adjusted prevalence ratio is the prevalence ratio adjusted for patients' gender, age, frequency of attending a GP and self-rated health and patient clustering. ¶ Fully adjusted prevalence ratio is the prevalence ratio adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. ** 95% confidence interval for the fully adjusted prevalence ratio. † † Poisson regression with robust variance ‡ ‡ Seniority in present practice § § "Single" = single-haned, "Shared single" = two ore more GPs sharing a single-handed practice, "Group of singles" = single-handed GPs sharing premises and staff, "Partnership" = GPs sharing patient-list, premises and staff, "Part-time partnership" = partnership practices with all GPs working part-time |||| Number converted to full-time working employees Prevalence ratio (PR) for 100% positive assessments and for 0-49% positive assessments associated with different patient characteristics. Crude PRs are adjusted for patient clustering. Patient-adjusted PRs are adjusted for patients' gender, age, frequency of attending a general practitioner (GP) and self-rated health and patient clustering. Fully adjusted PRs are adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. The goodness of model fitting is shown regarding the fully adjusted PRs (p-values). * Patients who marked 100% of the answered questions in one of the two most positive answering categories. † Patients who marked less than 50% (0-49%) of the answered questions in one of the two most positive answering categories. ‡ Prevalence § Crude prevalence ratio is the unadjusted prevalence ratio adjusted for patient clustering || Patient-adjusted prevalence ratio is the prevalence ratio adjusted for patients' gender, age, frequency of attending a GP and self-rated health and patient clustering. ¶ Fully adjusted prevalence ratio is the prevalence ratio adjusted for GPs' gender, age, weekly working hours, organisation of practice and staff in addition to patients' gender, age, frequency of attending a GP, self-rated health and patient clustering. ** 95% confidence interval for the fully adjusted prevalence ratio. † † Poisson regression with robust variance ‡ ‡ Seniority in present practice § § "Single" = single-haned, "Shared single" = two ore more GPs sharing a single-handed practice, "Group of singles" = single-handed GPs sharing premises and staff, "Partnership" = GPs sharing patient-list, premises and staff, "Part-time partnership" = partnership practices with all GPs working part-time |||| Number converted to full-time working employees # Discussion ## Principal findings We found a strong negative association between the GP's age and the evaluation of all aspects, except accessibility. We also found a strong association between the way the practice was organised and the patients' evaluation of accessibility, with GPs in single-handed practices getting far the most positive evaluations. Long weekly working hours were associated with more positive evaluations of all dimensions, except accessibility, whereas more than 0.5 full-time employees per GP, a comparatively high number of listed patients per GP and working in a training practice were associated with negative evaluation of accessibility. # Discussion of methods We chose to include the GPs' age as ten-year categories, and weekly working hours were categorised into two categories above and two categories below standard Danish full-time hours. For both variables we found general trends in their associations with assessments, which indicates that more narrow ranges would hardly have revealed additional patterns of associations. Seniority in present practice was preferred to seniority as a GP as the latter to some extent would correlate with the GP's age, while the former indicates the GP's opportunity to provide continuity. We did not include locums and vocational trainees in the variable "number of GPs sharing premises", even though they may have influenced the total practice resources and the number of staff needed. We chose a one-level model to account for patient clustering. However, GPs were also clustered in practices and a two-level model may therefore have been more applicable. Due to difficulties in converging the two-level model, we tested the differences between one-and two-level models and found that the variances between GPs were small, and differences in PR estimates were therefore negligible (below 2%). Overall, the model fit showed to be good using the Bernoulli family and in the situations where the Poisson regression was used. As earlier explained in the methods section, the high prevalences forced us to use Poisson regression in some instances to enable the model to converge, which in this study did not change the estimates [bib_ref] Alternatives for logistic regression in cross-sectional studies: an empirical comparison of models..., Barros [/bib_ref] [bib_ref] A modified poisson regression approach to prospective studies with binary data, Zou [/bib_ref]. # Strengths The study enjoyed a very high statistical precision, which meant that we were able to detect quite small statistically significant associations and one might question their clinical relevance. However, the precision was, indeed, con-siderable, so the risk of overlooking associations (type IIerror) was accordingly extremely low. In several cases, we found that GP and practice characteristics were differently associated with the evaluation of various aspects of care. Such differences may be blurred in measures of general satisfaction, which makes it difficult to compare our results with results from studies using this kind of measure [bib_ref] Patient satisfaction as an indicator of quality care, Cleary [/bib_ref] [bib_ref] Patient satisfaction: a review of issues and concepts, Sitzia [/bib_ref]. ## Weaknesses The project was a part of a large national patient evaluation project. This may have introduced some methodological weaknesses in relation to the aim of this particular study. All GPs in the involved counties were invited, but those who entered the project were not necessarily a representative fraction. The method of patient inclusion should ideally secure a random draw from the attending part of the listed patients, well knowing that frequently attending patients would be overrepresented. We do not know to which extent GPs forgot to hand out questionnaires or more systematically left some patients out. We focused on adjusted associations between evaluations and GP and practice characteristics. Selection of GPs and patients therefore did not have the same impact as if the aim had been to investigate actual evaluation levels. # Discussion of results Our finding that GP gender was not associated with the evaluation is in agreement with earlier findings [bib_ref] Characteristics of practices, general practitioners and patients related to levels of patients'..., Baker [/bib_ref] [bib_ref] Practice patterns, physicians' characteristics and patient-evaluated quality of general practice in Norway, Kvamme [/bib_ref] , whereas the statistically significantly more positive evaluation of the youngest GPs in this field represents new knowledge. Earlier studies have reported a positive association between patients' evaluation of general practice care and the GP's age [bib_ref] What type of general practice do patients prefer? Exploration of practice characteristics..., Baker [/bib_ref] , but negative associations between the evaluation and the GP's age and seniority have also been found [bib_ref] Characteristics of practices, general practitioners and patients related to levels of patients'..., Baker [/bib_ref] [bib_ref] Practice patterns, physicians' characteristics and patient-evaluated quality of general practice in Norway, Kvamme [/bib_ref]. We found no correlation between the GP's age and the number of patients per GP or practice organisation that could explain the association found. The negative association between GP age and the evaluation may indicate that patients experience younger GPs as more skilled or it may reflect possible deficiencies in the continuous medical education (CME) of older GPs. Maybe, patients expect more from older GPs or, maybe, GPs over time adjust their effort in order to counter burnout. We would have expected the impact of continuity in the relationship to be reflected in a positive association between GP age and the evaluation score. Maybe, a "shift of scenes" is sometimes appreciated by the patients. If the GP is not aware of this potential problem, continuity may imply a degree of preoccupancy that hinders him from perceiving and adjusting to changes in the patient's life situation and health. This challenges the perception of personal, continuous care as an unconditionally valuable quality [bib_ref] Saying 'goodbye' to single-handed practices; what do patients and staff lose or..., Van Den Hombergh [/bib_ref] [bib_ref] Continuity of care in general practice: effect on patient satisfaction, Hjortdahl [/bib_ref] ]. Yet another possible explanation may be that the association originated in a cohort effect, reflecting improvement in the CME of GPs, including both technical and inter-personal competencies. The GP's weekly working hours were not associated with the patients' perception of accessibility, but were positively associated with aspects of care organisation, including continuity, presumably because continuity is closely related to the GP personally, whereas lack of accessibility may be compensated for by the colleagues or managed otherwise. The geographical location of the practice was not associated with the evaluation. It is surprising, though, to see that practising in rural districts was not negatively associated with the evaluation of accessibility, even though regions with shortage of GPs were included in this study. This may possibly be explained by an adjustment of patient expectations due to public discussion of the problem. Like in earlier studies [bib_ref] What type of general practice do patients prefer? Exploration of practice characteristics..., Baker [/bib_ref] [bib_ref] Characteristics of practices, general practitioners and patients related to levels of patients'..., Baker [/bib_ref] [bib_ref] Practice size: impact on consultation length, workload, and patient assessment of care, Campbell [/bib_ref] [bib_ref] Identifying predictors of high quality care in English general practice: observational study, Campbell [/bib_ref] , the number of patients per GP was not consistently associated with the evaluation in the first four dimensions, but we were not surprised to see that it was negatively associated with the patients' perception of accessibility. Inversely, the number of staff was also negatively associated with accessibility. In Denmark, practice nurses and laboratory assistants can conduct consultations on their own if supervised by the GP. Delegating certain services to practice staff may induce a sense of limited access to the GP, which is supported by the fact that we found that GPs with more employees served larger lists of patients. In concordance with earlier findings [bib_ref] Practice size: impact on consultation length, workload, and patient assessment of care, Campbell [/bib_ref] [bib_ref] General practice in Gloucestershire, Avon and Somerset: explaining variations in standards, Baker [/bib_ref] , there was a slight tendency towards more positive evaluation of the GP's technical care in part-time partnership practices, but otherwise practice organisation mainly influenced the patients' experience of accessibility. GPs in single-handed practices got, by far, the most positive assessments of all, while part-time partnership practices obtained the most negative assessments. This supports results from earlier studies [bib_ref] Practice size: impact on consultation length, workload, and patient assessment of care, Campbell [/bib_ref] [bib_ref] Saying 'goodbye' to single-handed practices; what do patients and staff lose or..., Van Den Hombergh [/bib_ref]. More modest patient expectations of the single-handed GP's accessibility may explain this, or the explanation may be concealed by other factors regarding organisation, service and priorities that we have not been able to adjust for. Baker [bib_ref] What type of general practice do patients prefer? Exploration of practice characteristics..., Baker [/bib_ref] [bib_ref] Characteristics of practices, general practitioners and patients related to levels of patients'..., Baker [/bib_ref] found training practices to obtain less favourable assessments than non-training practices. Being involved with teaching and training out of practice was not associated with the evaluation in our study and working in a training practice was only associated with a comparatively lower evaluation of accessibility. ## Policy and practice implications Our results suggest adjustment for GP age whenever evaluation results are compared between practices. On the other hand, both the GPs themselves and policy makers must be aware of correctable consequences of increasing GP age. Depending on the situation, adjustment for practice organisation may also be appropriate, but as practice organisation may be closely interwoven with other organisational aspects, this may blur important differences between practices that ought to be addressed. We found single-handed practice, a short patient list, only a few employees, and not being a training practice to be associated with a better patient-experienced accessibility to care. This is contradictory to the finding of better technical performance of GPs in larger partnerships [bib_ref] Practice size: impact on consultation length, workload, and patient assessment of care, Campbell [/bib_ref] [bib_ref] General practice in Gloucestershire, Avon and Somerset: explaining variations in standards, Baker [/bib_ref] , to the current trend in society that favours larger primary care units and that younger doctors wish to work parttime hours in partnership practices. Further studies are therefore needed to discover what lies behind these results and how practice may be organised in order to secure high professional standards and at the same time satisfy the patients' needs for personal continuity and accessibility. As this is in some way a general paradox concerning all practices, policy makers and GPs should come together to seek for possible solutions to this problem. It cannot be concluded that patient evaluation results should always or never be adjusted for differences between practices. Adjustment may be appropriate when comparing results between (groups of) practices, but in the perspective of individual practices, one may claim that every GP must strive to satisfy his patients disregarding that his working conditions may be different from those of his colleagues. # Conclusion We found the GP's age to be negatively associated with the patients' evaluation of all aspects of care, except accessibility. We also found a strong association between the way the practice was organised and the patients' evaluation of accessibility, with GPs in single-handed practices getting far the most positive evaluations. We suggest that future evaluations be adjusted for differences in the GPs' age and practice organisation before comparing (groups of) practices. Long weekly working hours were associated with more positive evaluations of all dimensions, except accessibility, whereas more than 0.5 full-time employees per GP, a comparatively high number of listed patients per GP and working in a training practice were associated with negative evaluation of accessibility. These results may be used to adjust practice in order to increase the patientexperienced accessibility to care. [table] Table 1: Distribution of characteristics among participating general practitioners. [/table] [table] Table 2: Distribution of characteristics among participating patients. [/table] [table] Table 3: Dimension 1: Crude and adjusted associations between patients' evaluation of aspects of the doctor-patient-relationship. [/table] [table] Table 4: Dimension 2: Crude and adjusted associations between patients' evaluation of aspects of the medical care. Prev ‡ (%) Crude PR § Pt-adj. PR || Adj. PR ¶ Adj. 95%CI** Prev ‡ (%) Crude PR § Pt-adj. PR || Adj. PR ¶ [/table] [table] Table 5: Dimension 3: Crude and adjusted associations between patients' evaluation of aspects of the information and support. Prev ‡ (%) Crude PR § Pt-adj. PR || Adj. PR ¶ Adj. 95%CI** Prev ‡ (%) Crude PR § Pt-adj. PR || Adj. PR ¶ [/table] [table] Table 6: Dimension 4: Crude and adjusted associations between patients' evaluation of aspects of the organisation of care. [/table] [table] Table 7: Dimension 5: Crude and adjusted associations between patients' evaluation of aspects of the accessibility. [/table]

Same features, different diagnosis: A case of ilio-iliac arteriovenous fistula presenting as decompensated heart failure LessonIn case of high output cardiac failure, an arteriovenous fistula should be considered. High-output heart failure due to ilio-iliac arteriovenous fistula is a rare phenomenon. We describe a case where patient initially presented with features of heart failure. He had a history of dilated cardiomyopathy and cardiac ablation procedure done for atrial flutter. Subsequent decompensation and multiorgan failure led to extensive diagnostic work-up revealing a normal heart and an arteriovenous fistula that was managed successfully with endovascular repair. ## Case presentation A 58-year-old driving instructor presented with a fourweek history of worsening breathlessness, abdominal distension and leg swelling. He had presented with similar features about six years ago. He was then diagnosed with alcohol-related dilated cardiomyopathy with impaired left ventricular systolic function (ejection fraction 25%) as he was then drinking over 120 units of alcohol a week and had a normal coronary angiogram. He had an atrial ablation procedure for atrial flutter four years ago and following a period of abstinence his heart failure symptoms had resolved. He also had a history of hypertension and recently diagnosed with type 2 diabetes. He had started drinking moderate amounts of alcohol again. On examination, he had a heart rate of 92 and blood pressure of 128/54. He had 94% oxygen saturation on pulse oximetry on air with a normal respiratory rate. He had an elevated jugular venous pressure up to his right ear lobe. Auscultation of heart and lungs revealed a loud pan-systolic murmur and bi-basal crepitations. His abdomen was markedly distended with 5 cm non-tender hepatomegaly and shifting dullness. He had significant peripheral oedema up to his umbilicus. Chest X-ray showed pulmonary congestion. Electrocardiography on admission showed sinus rhythm with no ischaemic changes and no evidence of left ventricular hypertrophy. Blood tests on admission were normal other than a slightly elevated international normalised ratio (1.5). The patient was initially diagnosed and managed as decompensated heart failure. The patient's oedema and ascites did not improve with diuretics and his weight increased from 128 kg to 142 kg. His renal and liver functions deteriorated and he became increasingly jaundiced (bilirubin 83, aspartate aminotransferase 188, alkaline phosphatase 179, international normalised ratio 2.6). An up-to-date echocardiogram showed only mild left ventricular dilation with normal left ventricle and right ventricle function (left ventricular ejection fraction 60%). A liver ultrasound scan confirmed hepatomegaly and showed a dilated portal vein and evidence of periportal fibrosis. With a working diagnosis of liver cirrhosis, a computed tomography scan was arranged that did not show any radiological evidence of cirrhosis. After discussion at the hepatology multidisciplinary meeting, it was decided to perform a transjugular biopsy of the liver to rule out cirrhosis and paracentesis to relieve his worsening ascites. Transjugular biopsy showed greatly elevated systemic venous pressures (inferior vena cava pressure: 40 mmHg, normal 5-10 mmHg) and elevated portal pressures (wedged portal pressure: 45 mmHg, normal range 1-5 mmHg) but a normal hepatic venous pressure gradient (normal range < 5 mmHg); findings consistent with systemic venous hypertension rather than portal hypertension. An ultrasound scan guided paracentesis drained 8 L of ascitic fluid. After excluding cirrhosis, other causes of hepatorenal dysfunction were considered as the patient was progressively getting worse. He eventually started complaining of pain and paraesthesia in his lower limbs. Acute limb ischaemia was suspected and urgent duplex ultrasound scan arranged. Duplex scan showed poor distal flow in both lower limbs. A large right sided common iliac aneurysm was noted with turbulent arterial flow and highly phasic flow in the common femoral vein suggestive of an arteriovenous fistula. These findings were confirmed with computed tomographic angiogram [fig_ref] Figure 2: Angiogram with contrast being injected through left groin [/fig_ref]. The patient was urgently transferred to the regional vascular surgery unit where he underwent endovascular repair. A satisfactory exclusion of the fistula was achieved with placement of a stent graft from just below the renal artery to both iliac bifurcations [fig_ref] Figure 4: A completion angiogram showing exclusion of fistula by the stent graft [/fig_ref]. There was an immediate improvement with reduction in his central venous pressure and improvement in urine output after the repair. Four days after the procedure (first two days in high dependency unit), patient was feeling much better and renal and liver function was markedly improved. He was discharged home nine days after the operation. He was reviewed in outpatients after two weeks and was significantly better clinically with no requirements for any antihypertensive or diabetic medicines. His liver biopsy showed changes consistent with hepatic congestion (cardiac cirrhosis) but confirmed there was no evidence of alcohol-induced liver injury. # Discussion An ilio-iliac arteriovenous fistula presenting with decompensated heart failure, ascites and deranged liver and kidney function is rare. 1,2 This case was more unusual as the patient had a previous history of heart failure attributed to alcohol-related dilated cardiomyopathy that recovered completely after a period of alcohol abstinence. Causes and presenting features of an arteriovenous fistula are quite varying and depend on their location, size, duration, calibre of vessels and the cardiopulmonary reserve of the patient. [bib_ref] Aortocaval and iliac arteriovenous fistulas: recognition and treatment, Brewster [/bib_ref] An ilio-iliac arteriovenous fistula is a rare type of extra splanchnic abdominal arteriovenous fistula. Causes include aneurysmal rupture (spontaneous), trauma, surgery (iatrogenic), malignancy and infection. Cardiac catheterisation procedures are a known cause of groin arteriovenous fistula and the incidence reported is less than 1%. [bib_ref] A prospective study on incidence and risk factors of arteriovenous fistulae following..., Perings [/bib_ref] Patients are classically reported to have high output heart failure, abdominal and/or lower back pain, palpable abdominal lump with a bruit and signs of local venous hypertension, i.e. lower extremity oedema, haematuria. [bib_ref] The syndrome of spontaneous iliac arteriovenous fistula: a distinct clinical and pathophysiologic..., Mcauley [/bib_ref] These features, however, were found in only 20-50% of patients in one reported study. [bib_ref] Aortocaval and iliac arteriovenous fistulas: recognition and treatment, Brewster [/bib_ref] Highoutput heart failure is characterised by cardiac index of more than 4 L/min per m 2 . It is more common, present in 35% to 58% of cases, in aortoiliac arteriovenous fistula due to the large calibre of vessels. [bib_ref] High-output heart failure: how to define it, when to treat it, and..., Wasse [/bib_ref] An arteriovenous fistula causes haemodynamic instability by increasing cardiac output as a result of shunting blood from high resistance arterial flow to low resistance venous circulation. It increases blood volume and heart rate and contractility by affecting the sympathetic and renin-angiotensin-aldosterone system (RAAS). One study describes an increase of 10% in cardiac output within two weeks of making an arteriovenous fistula fistula. [bib_ref] Effects of arteriovenous fistula formation on arterial stiffness and cardiovascular performance and..., Korsheed [/bib_ref] Arteriovenous fistulae, as in this case, can lead to multiorgan failure associated with increased morbidity and mortality. Surgical mortality in symptomatic arteriovenous fistula can be as high as 22-51%. [bib_ref] Diagnosis of an ilio-iliac arteriovenous fistula by multidetector row computed tomography and..., Goto [/bib_ref] Mortality is potentially higher in patients without a preoperative diagnosis of such fistula. [bib_ref] Aortocaval and iliac arteriovenous fistulas: recognition and treatment, Brewster [/bib_ref] The diagnosis remains unestablished in almost half the cases of Iliac and aortic arteriovenous fistulae. [bib_ref] Diagnosis of an ilio-iliac arteriovenous fistula by multidetector row computed tomography and..., Goto [/bib_ref] [bib_ref] Successful surgical repair of an ilio-iliac arteriovenous fistula associated with a ruptured..., Iijima [/bib_ref] This is probably due to unusual presentations of these patients going through a few working diagnoses before the final diagnosis of arteriovenous fistula and acquiring a presumed clinical urgency by then. Abdominal bruit was the most consistent feature in such patients and yet was frequently overlooked. [bib_ref] Aortocaval and iliac arteriovenous fistulas: recognition and treatment, Brewster [/bib_ref] Contrast-enhanced computed tomography or an angiography is the most commonly utilised diagnostic modality. Principles of fistula repair can be achieved by both open and endovascular techniques. Endovascular repair has shown promising results with benefits of being less invasive and causing less risk of major blood loss as shown in our case. 10 . Post-endovascular repair. Stent grafts inserted from just below renal arteries to bifurcation of right common iliac artery to exclude the fistula. Empty aneurysmal sac can also be seen. Ethical approval: Written informed consent for the publication was obtained from the patient. # Declarations Guarantor: SR. Contributorship: SR: Conception and design of article; analysis and/or interpretation of data; literature search; drafting the manuscript; revising the manuscript critically for important intellectual content; manuscript approval. HS: Acquisition of data; drafting the manuscript; revising the manuscript critically for important intellectual content; manuscript approval. AR: Analysis and/or interpretation of data; manuscript revision; manuscript approval. AK: Conception and design of article; literature search; manuscript approval. KW-P: Revising the manuscript critically for important intellectual content; manuscript approval. SWY: Conception and design of article; provision of radiology images; revising the manuscript critically for important intellectual content; manuscript approval. [fig] Figure 2: Angiogram with contrast being injected through left groin: Right Common Iliac artery aneurysm and fistula. Note markedly dilated inferior vena cava. [/fig] [fig] Figure 1: Coronal view of right common iliac artery aneurysm and fistula between it and right common iliac vein. Note the significantly dilated inferior vena cava and hepatic veins. [/fig] [fig] Figure 4: A completion angiogram showing exclusion of fistula by the stent graft. [/fig] [fig] Figure 3: Post-endovascular repair. Stent grafts inserted from just below renal arteries to bifurcation of right common iliac artery to exclude the fistula. Empty aneurysmal sac can also be seen. [/fig]

Couples HIV counselling and couple relationships in India, Georgia and the Dominican Republic Background: Couples HIV counseling and testing is essential for combination HIV prevention, but its uptake remains very low. We aimed to evaluate factors associated with couples HIV counseling uptake in India, Georgia and the Dominican Republic, as part of the ANRS 12127 Prenahtest intervention trial. Methods: Pregnant women ≥15 years, attending their first antenatal care (ANC) session between March and September 2009, self-reporting a stable partner, and having received couple-oriented post-test HIV counseling (trial intervention) were included. Individuals and couple characteristics associated with the acceptability of couples HIV counseling were assessed using multivariable logistic regression for each study site.Results: Among 711 women included (232, 240 and 239 in the Dominican Republic, Georgia and India, respectively), the uptake of couples HIV counseling was 9.1% in the Dominican Republic, 13.8% in Georgia and 36.8% in India. The uptake of couples HIV counseling was associated with women having been accompanied by their partner to ANC, and never having used a condom with their partner in the Dominican Republic; with women having been accompanied by their partner to ANC in India; with women having a higher educational level than their partner and having ever discussed HIV with their partner in Georgia.Conclusion: Couple HIV counseling uptake was overall low. Strategies adapted to local socio-cultural contexts, aiming at improving women's education level, or tackling gender norms to facilitate the presence of men in reproductive health services, should be considered. Trial registration: ClinicalTrials.gov Identifier: NCT01494961. Registered December 15, 2011. (Retrospectively registered). # Background Worldwide, an estimated 2.1 million people became newly infected with HIV infection in 2015. A very large part of these new infections occurred within heterosexual and serodiscordant stable couple relationships [bib_ref] Status in discordant couples in sub-Saharan Africa: a systematic review and metaanalysis, Eyawo [/bib_ref] , as evidenced by the abundant literature from sub-Saharan Africa [bib_ref] Sources of HIV incidence among stable couples in sub-Saharan Africa, Chemaitelly [/bib_ref] [bib_ref] Incidence of HIV infection in stable sexual partnerships: a retrospective cohort study..., Hugonnet [/bib_ref] [bib_ref] Rates of HIV-1 transmission within marriage in rural Uganda in relation to..., Carpenter [/bib_ref] [bib_ref] Couples at risk: HIV-1 concordance and discordance among sexual partners receiving voluntary..., Malamba [/bib_ref]. Several Asian countries have also reported high levels of intimate partner transmission of HIV, with women living with HIV being infected by their married partner who engaged in unsafe behaviors, prior or after marriage [bib_ref] Marriage, monogamy and HIV: a profile of HIV-infected women in south India, Newmann [/bib_ref]. Preventing HIV transmission among serodiscordant couples thus remains a key target in the worldwide fight against HIV. Significant new advances have taken place in the past years in the field of HIV prevention research, mainly in the field of biomedical interventions for serodiscordant couples. The HPTN052 trial showed that when an HIVinfected person is on antiretroviral (ARV) treatment (ART) with good adherence, his/her risk of HIV transmission to his/her uninfected partner is significantly decreased [bib_ref] Prevention of HIV-1 infection with early antiretroviral therapy, Cohen [/bib_ref]. Within the PARTNER Pre-exposure Prophylaxis (PreP) study, when the HIV-negative partner used ARV drugs as prophylaxis, his/her risk of HIV acquisition from the HIV-infected partner was significantly reduced [bib_ref] Antiretroviral prophylaxis for HIV prevention in heterosexual men and women, Baeten [/bib_ref]. This evidence contributed to the revision of the World Health Organization guidelines in September 2015, which recommend initiation of ART to all HIV-infected people regardless of their immunological status, as well as ARVs as prophylaxis for serodiscordant couples. Implementing biomedical interventions aiming at preventing HIV acquisition and transmission within couples requires a first critical step, i.e. HIV status awareness within couples. Couple members who get tested together and mutually disclose their HIV status are more likely to adopt HIV prevention behaviors, both in HIV concordant or discordant relationships [bib_ref] Sexual behavior of HIV discordant couples after HIV counseling and testing, Allen [/bib_ref] [bib_ref] Eyes that don't see, heart that doesn't feel: coping with sex work..., Syvertsen [/bib_ref] [bib_ref] Effect of serotesting with counselling on condom use and seroconversion among HIV..., Allen [/bib_ref] [bib_ref] Couplebased HIV. Counseling and testing: a risk reduction intervention for US drug-involved..., Mcmahon [/bib_ref] [bib_ref] Couple-based behavioral HIV interventions: placing HIV risk-reduction responsibility and agency on the..., El-Bassel [/bib_ref] [bib_ref] Effectiveness of couple-based HIV counseling and testing for women substance users and..., Mcmahon [/bib_ref]. Further, women who receive prenatal HIV counseling along with the partner are more likely to adhere to prevention of mother-to-child transmission (PMTCT) interventions when they test HIVpositive than those who are counseled individually [bib_ref] Maximizing the impact of HIV prevention efforts: interventions for couples, Medley [/bib_ref]. Couples HIV counseling and testing has been encouraged by international agencies for many years, but efforts have mainly been restricted to the context of generalized HIV epidemics in sub-Saharan Africa [bib_ref] Implementation of couples' voluntary HIV counseling and testing services in Durban, South..., Kilembe [/bib_ref] [bib_ref] Official invitation letters to promote male partner attendance and couple voluntary HIV..., Jefferys [/bib_ref]. Extensive research has been conducted on couples HIV counseling in Rwanda and Zambia, including on promotion strategies [bib_ref] Promotion of couples' voluntary counselling and testing for HIV through influential networks..., Allen [/bib_ref] [bib_ref] Promotion of couples' voluntary HIV counselling and testing in Lusaka, Zambia by..., Wall [/bib_ref] and on the profile of couples accepting such servicesin Zambia, having been previously tested for HIV and cohabiting were among the main factors associated with couples HIV counseling uptake [bib_ref] Promotion of couples' voluntary HIV counselling and testing in Lusaka, Zambia by..., Wall [/bib_ref]. In Zambia, couples HIV counseling was shown to be associated with sustained reductions in selfreported unprotected sex [bib_ref] Sustained effect of couples' HIV counselling and testing on risk reduction among..., Wall [/bib_ref]. High rates of couples HIV counseling were recently reported at national level in Rwanda, preventing an estimated >70% of incident HIV infections [bib_ref] Implementation and Operational Research: Evolution of Couples' Voluntary Counseling and Testing for..., Karita [/bib_ref]. In most countries however, the proportion of couples who test together in the context of pregnancy is less than 20%, and the provision of couple HIV counseling and testing services is low [bib_ref] Systematic review of couple-based HIV intervention and prevention studies: advantages, gaps, and..., Jiwatram-Negrón [/bib_ref]. There is little data on the uptake of couples HIV counseling and testing in low to medium HIV prevalence countries and outside sub-Saharan Africa. And yet in such settings, low coverage of HIV testing clearly represents missed opportunities for preventing primary acquisition of HIV within couples. Further, the role of different types of couple relationships and gender norms, and of the local social and epidemiological contexts, on the acceptability of couple approaches to HIV prevention is poorly understood. This paper aims to describe the uptake of and factors associated with couples HIV counseling in low HIV prevalence settings. # Methods ## Study setting: the 12,127 prenahtest trial We conducted this analysis using the data collected in the ANRS 12127 Prenahtest study, a multicenter randomized controlled trial carried out between 2009 and 2011 in four urban health facilities located in Yaoundé (Cameroon), Santo Domingo (The Dominican Republic), Tbilisi (Georgia) and Pune (Maharashtra province, India). In 2014, HIV prevalence among adults aged 15-49 was estimated at 0.3% in Georgia, 0.4% in Maharashtra province, 0.8% in the Dominican Republic in 2014 and 4.8% in Cameroon. Trial methods were described previously [bib_ref] Increasing HIV testing among male partners, Orne-Gliemann [/bib_ref]. In summary, the trial was designed to evaluate the acceptability, feasibility and impact of an innovative prenatal counseling intervention offered to pregnant women, named Couple-Oriented post-test HIV Counseling (COC). The main study outcomes were the frequency of men's HIV testing, couples HIV counseling and couples HIV testing, as well as sexual, reproductive, and HIV prevention behaviors. Pregnant women were recruited during antenatal care (ANC). The trial baseline criteria were acceptance of trial participation, being aged 15 years or older, attending their first ANC visit in the four study centers between March and September 2009, no previous HIV test during their current pregnancy, and self-reporting having a stable partner. Women who were unable to provide consent due to mental illness at baseline, or those whose partner was absent for more than six months a year or whose partner had been tested for HIV during the current pregnancy were not eligible. Enrolled women were randomized to receive either Standard post-test HIV Counseling (SC arm) or the COC intervention (COC arm) [bib_ref] Implementation and Operational Research: Evolution of Couples' Voluntary Counseling and Testing for..., Karita [/bib_ref]. Specifically trained HIV counselors delivered the COC intervention. COC sessions included role-play to develop women's communication skills, especially to discuss HIV and sexual issues with their partner. Women were also encouraged to invite their partner for HIV testing [bib_ref] Increasing HIV testing among male partners, Orne-Gliemann [/bib_ref] and couples HIV counseling. All women responded to three face-to-face questionnaires: at baseline, before HIV testing (T0), within two to eight weeks after post-test HIV counseling (T1) and six months post-partum (T2). ## Study population This analysis is restricted to the three low HIV prevalence countries (The Dominican Republic, Georgia and India) and to women randomized in the COC group. We excluded women who had missing data for study variables. ## Variables of interest Our outcome of interest was the uptake of couples HIV counseling at the end of the trial, defined as pre-test HIV counseling received together by both partners (even if the couple did not receive couples HIV testing or couples post-test HIV counseling). It was measured from two main sources: i/women self-reports within the questionnaires administered at T1 and T2 and ii/ a trial impact form in which all events of interest for the trial (partner HIV testing and couples HIV counseling in particular) were notified by local trial coordinators. We assumed that women not seen at T1 or T2 and without any information in the trial impact form did not receive couples HIV counseling during the study period. Individual and couple-related characteristics were included in our analysis. Individual characteristics included age and educational level of the pregnant woman, women's HIV testing history and reported partner's HIV testing history. Couple-related characteristics included age and educational difference between the two partners, remunerated activity in the couple, cohabitation, duration of the relationship, report to be accompanied by partner to ANC, perception of partner support for pregnancy, report of emotional, verbal abuse or physical abuse at least once in the last month, and report of past discussion within the couple about HIV and condom use. # Statistical analysis We described the uptake of couples HIV counseling at the end of the study for each study site/country. Individual and couple characteristics at baseline were described in terms of numbers and frequencies. Unadjusted (univariable) and multivariable logistic regression models for each study site were used to assess the association between the uptake of couples HIV counseling and the individual and couple-related characteristics. We used a likelihood ratio test (LRT) to screen for variables to be included in the adjusted (multivariable) analyses. Factors that were significant at P < 0.25 in unadjusted analyses and woman age as confounder were included in the multivariable model. Final models were retained using a backward elimination approach and after checking for confounding factors. Analyses were carried out using SAS version 9.3. # Ethics statement The Prenahtest study was approved by Comité de Ética Indepediente, Fundación Dominicana de Infectología (9 April 2007, Dominican Republic), IRB 00006752 of Maternal and Child Care Union (13 November 2008, Georgia), Independent Ethics Committee for Prayas Health Group . Participants were assigned identification numbers and all questionnaires and process forms were labeled with matching numbers to maintain confidentiality. # Results ## Description of the study population Overall, 1459 pregnant women were recruited and randomized in the three Prenahtest trial study sites, and 1445 of them responded to the baseline questionnaire. We excluded 723 women randomized to the SC groups and 11 with missing data. A total of 711 pregnant women were included in the analysis, among which 232 in the Dominican Republic, 240 in Georgia and 239 in India [fig_ref] Figure 1: Selection of the study population [/fig_ref]. Individual and couple characteristics at baseline are described in [fig_ref] Table 1: Baseline characteristics of women who received couple-oriented post-test HIV counseling [/fig_ref]. Dominican and Indian women were aged 21 years in median (interquartile range (IQR) = [bib_ref] Implementation of couples' voluntary HIV counseling and testing services in Durban, South..., Kilembe [/bib_ref] [bib_ref] Official invitation letters to promote male partner attendance and couple voluntary HIV..., Jefferys [/bib_ref] [bib_ref] Promotion of couples' voluntary counselling and testing for HIV through influential networks..., Allen [/bib_ref] [bib_ref] Promotion of couples' voluntary HIV counselling and testing in Lusaka, Zambia by..., Wall [/bib_ref] [bib_ref] Sustained effect of couples' HIV counselling and testing on risk reduction among..., Wall [/bib_ref] [bib_ref] Implementation and Operational Research: Evolution of Couples' Voluntary Counseling and Testing for..., Karita [/bib_ref] [bib_ref] Systematic review of couple-based HIV intervention and prevention studies: advantages, gaps, and..., Jiwatram-Negrón [/bib_ref] and IQR = [bib_ref] Implementation of couples' voluntary HIV counseling and testing services in Durban, South..., Kilembe [/bib_ref] [bib_ref] Official invitation letters to promote male partner attendance and couple voluntary HIV..., Jefferys [/bib_ref] [bib_ref] Promotion of couples' voluntary counselling and testing for HIV through influential networks..., Allen [/bib_ref] [bib_ref] Promotion of couples' voluntary HIV counselling and testing in Lusaka, Zambia by..., Wall [/bib_ref] , respectively) and most of them had completed less than 13 years of education (91.5% and 87.5%, respectively). Georgian women were aged 25 years in median (IQR = [bib_ref] Promotion of couples' voluntary counselling and testing for HIV through influential networks..., Allen [/bib_ref] [bib_ref] Promotion of couples' voluntary HIV counselling and testing in Lusaka, Zambia by..., Wall [/bib_ref] [bib_ref] Sustained effect of couples' HIV counselling and testing on risk reduction among..., Wall [/bib_ref] [bib_ref] Implementation and Operational Research: Evolution of Couples' Voluntary Counseling and Testing for..., Karita [/bib_ref] [bib_ref] Systematic review of couple-based HIV intervention and prevention studies: advantages, gaps, and..., Jiwatram-Negrón [/bib_ref] [bib_ref] Increasing HIV testing among male partners, Orne-Gliemann [/bib_ref] [bib_ref] Effectiveness of antenatal group HIV voluntary counseling and testing services in rural..., Gupta [/bib_ref] and most of them (70.8%) had completed at least 13 years of education. In Georgia, 80% of women reported being in a married relationship. All women enrolled in India were married. In the Dominican Republic, >84% reported living in free union (i.e. living with a partner without legal or religious recognition). Women often lived with both their partner and his family members in Georgia (45%) and India (52%), while in Dominican Republic women were more likely to live with their partner only (i.e. as a nuclear family) (76.7%). In all three sites, less than one third of pregnant women were employed at the time of enrolment and up to 20% of both partners were not working in Georgia. Few women had a higher educational level than their partner (respectively 24%, 29% and 39% of women in Georgia, India and the Dominican Republic). In all three countries, women had occasionally been accompanied by their partner to their first ANC visit: 44% in India, 38% in Georgia and 28% in the Dominican Republic. Approximately two thirds of women in the Dominican Republic and India perceived their partner as very supportive of their pregnancy. 48.3% of women in the Dominican Republic reported experiencing emotional or verbal abuse from their partner at least once in the last month prior enrolment. This proportion was lower in Georgia (25.8%) and India (19.7%). Physical abuse by the partner in the last month was reported by 16.3% of women in India, 10.3% in the Dominican Republic and 1.7% in Georgia. Frequent alcohol consumption by their partner was reported by 3.8% of women in the Dominican Republic, 6.9% in Georgia and 6.6% in India. Previous HIV testing was reported by 37.9% of women in Georgia, [bib_ref] Male perspectives on incorporating men into antenatal HIV counseling and testing, Katz [/bib_ref] ## Uptake of couples hiv counseling at the end of the trial Among the 711 women included in this analysis, 131 were seen at T1 only, 507 at T1 and T2, 11 at only T2 and 62 were not seen neither at T1 nor T2. A total of 141 women (19.8% of all included women) reported receiving couples HIV counseling during the trial, either at T1 (114 women), or at T2 (28 women). The uptake of couples HIV counseling was uneven by country: 9.1% (21 couples) in the Dominican Republic, 13.8% (33 couples) in Georgia and 36.8% (88 couples) in India [fig_ref] Figure 2: Uptake of couples HIV counseling among women who received couple-oriented post-test counseling [/fig_ref]. Individual and couples characteristics associated with the uptake of couples HIV counseling. and adjusted (multivariable) analyses in [fig_ref] Table 3: Multivariable analysis of factors associated with the uptake of couples HIV counseling [/fig_ref]. ## Unadjusted (univariable) analyses are presented in In Dominican Republic, the multivariable analysis showed that the uptake of couples HIV counseling was significantly more likely among women who were accompanied by their partner to their first ANC visit (17.7% versus 6.1%, adjusted Odds-Ratio (aOR) = 4.10, 95% Confidence Interval (95% CI) = [1.51, 11.13]). Couples HIV counseling was also more likely among women who had never used a condom with their partner but had ever discussed it (15.8%) and among those who had never used a condom and never discussed it with the partner (13.1%), compared to those who declared ever having used a condom and ever having discussed it with their partner (5.0%) (respectively, aOR = 6.17, 95% CI = [1.57, 24.20] and aOR = 3.67, 95% CI = [1.07, 12.63]). The uptake of couples HIV counseling was less likely among women who perceived their partner as normally or less supportive of the current pregnancy compared to those who perceived their partner as supportive (3.5% versus 12.3%, aOR = 0.26, 95% CI = [0.73-0.90]). . In Georgia, the multivariable analysis showed that the uptake of couples HIV counseling was significantly more likely among women with a higher educational level than their partner, compared to the contrary (27.6% versus 9.9%, aOR = 3.45, 95% CI = [1.56, 77]). The uptake of couples HIV counseling was less likely among women who had never discussed about HIV with their partner, compared to those who had had a discussion about HIV only on general terms (7.9% versus 20.7%, aOR = 0.33, 95% CI = [0.14, 0.72]) . In India, in the multivariable analysis, the uptake of couples HIV counseling was more likely among women who had been accompanied by their partner to their first ANC visit (52.4% versus 24.6%, OR = 3.37, 95% CI = [1.95, 5.83]) . # Discussion Our results showed that the uptake of couples HIV counseling within the Prenahtest trial, among women who specifically received an intervention dedicated to supporting a couples approach to HIV counseling and testing, was low in the Dominican Republic and Georgia (respectively 9.1% and 13.8%) and a little higher in India (36.8%). Further, the uptake of couples HIV counseling was overall lower than what we reported for partner HIV testing [bib_ref] Increasing HIV testing among male partners, Orne-Gliemann [/bib_ref] , highlighting the challenges of providing couples HIV services in antenatal settings. Our analysis also provided insight into the profile of women and their partner who engaged in couples HIV counseling, which varied substantially across study sites. In Georgia, couples in which the woman was more educated than her partner were more likely to receive couples HIV counseling. The role of education level on the acceptability and impact of behavioral interventions has been shown in several low HIV prevalence contexts, including in India [bib_ref] Effectiveness of antenatal group HIV voluntary counseling and testing services in rural..., Gupta [/bib_ref] [bib_ref] Epidemiologic correlates of willingness to be tested for HIV and prior testing..., Das [/bib_ref]. More educated woman may have had stronger confidence to initiate discussion about HIV and better negotiation skills within their couple relationship to encourage their partner to receive couples HIV counseling. Previous discussions about HIV within the couple also contributed to a better uptake of couples HIV counseling. Thus, promoting communication within couples and bringing information about HIV to both couple members might contribute to improve couples HIV counseling uptake in the Georgian context. In India, only one characteristic was associated with the uptake of couples HIV counseling: the fact that the woman was accompanied by the partner to ANC. The presence of men in ANC seems to have triggered some form of "provider-initiated couples HIV counseling", which proved highly effective. These findings are comparable with those recently reported in a pilot project in 15 hospitals in Thailand, with a very high uptake of couples HIV counseling among women accompanied by their partner to ANC [bib_ref] Assessment of a couples HIV counseling and testing program for pregnant women..., Lolekha [/bib_ref]. In the Dominican Republic, in addition to women being accompanied to ANC by their partner, women's perception of a strong involvement of their partner in the pregnancy was associated with the uptake of couples HIV counseling. This association was not found in the two others countries or in the literature. Also, couples that had never used a condom were more likely to receive couples HIV counseling than those who had already used condoms and discussed about it. These results may be explained by the fact that couples with little information on HIV might feel less anxious about HIV screening. This hypothesis is supported by the results of a study conducted in Kenya in 2001 that found that men who opted for couples HIV counseling had less knowledge about HIV than those who chose individual HIV counseling [bib_ref] Male perspectives on incorporating men into antenatal HIV counseling and testing, Katz [/bib_ref]. One of the limits of our study may be that we restricted our analysis to women from the intervention group. This choice was made based on methodological reasons, as in Georgia, no women from the SC group had reported receiving couples HIV counseling. The fact that we excluded one of the four study sites may have led to a partial appreciation of Prenahtest results, but this choice was motivated by epidemiological reasons, as Cameroon has a much higher HIV prevalence than the three other sites, with prevalence rates below 1%. Finally, the fact that all data were self-reported by women may have led in part to an information bias regarding partner and couple characteristics. Overall however, this multi-site analysis allowed investigating couples HIV counseling in three very different socio-demographic contexts, while ensuring a standardized approach in comparing results between sites. Our analysis added to the scarce literature on couples HIV prevention in low-income, low HIV prevalence and non-African cultural settings. These findings suggest overall that improving the uptake of couples HIV counseling will require substantial structural changes, such as improving women's education level or encouraging an evolution in gender norms to facilitate the presence of men in health services for sexual and reproductive health and to strengthen the ability of women to discuss HIV with their partners. The urgent need for an improved HIV response does not fit well with such longterm behavior change. However, the current changes in the HIV care landscape with the generalization of ART, which will impact on individual health, may sooner impact couple relationships and contribute to higher acceptability and uptake of biomedical and behavioral interventions that address couple concerns. # Conclusion Although the efficacy of the COC intervention tested within the Prenahtest trial was limited in terms of uptake of couples HIV counseling, we showed previously its value in improving partner HIV testing [bib_ref] Increasing HIV testing among male partners, Orne-Gliemann [/bib_ref] and short-term communication about HIV within couples [bib_ref] Enhanced prenatal HIV couple oriented counselling session and couple communication about HIV..., Plazy [/bib_ref]. Other couples-centered strategies have been evaluated to increase the uptake of HIV testing and the adoption of HIV prevention strategies within couples. These are essentially community-based interventions such as home-based visits [bib_ref] Effect of home based HIV counselling and testing intervention in rural South..., Doherty [/bib_ref] [bib_ref] Strong effects of home-based voluntary HIV counselling and testing on acceptance and..., Fylkesnes [/bib_ref] [bib_ref] Home visits during pregnancy enhance male partner HIV counselling and testing in..., Osoti [/bib_ref] , and clinic-based interventions such as invitation letters from health care workers to partners [bib_ref] The forgotten half of the equation: randomized controlled trial of a male..., Mohlala [/bib_ref]. The efficacy of women-delivered HIV self-test in reaching men, who remain underserved by existing services, is about to be evaluated [bib_ref] Acceptability of woman-delivered HIV self-testing to the male partner, and additional interventions:..., Choko [/bib_ref]. Combined strategies, tailored to the socio-cultural context, will be critical to improve the uptake of couples HIV counselling and testing and overall interventions for HIV prevention among couples in low to medium HIV prevalence countries. [fig] Figure 1: Selection of the study population. Prenahtest ANRS 12127 trial [/fig] [fig] Figure 2: Uptake of couples HIV counseling among women who received couple-oriented post-test counseling. Prenahtest ANRS 12127 trialTable 2Univariable analysis of factors associated with the uptake of couples HIV counseling. [/fig] [table] Table 1: Baseline characteristics of women who received couple-oriented post-test HIV counseling. Prenahtest ANRS 12127 trial [/table] [table] Table 3: Multivariable analysis of factors associated with the uptake of couples HIV counseling. Prenahtest ANRS 12127 trial Emotional or verbal abuse from partner at least once in the last month AOR Adjusted Odds-Ratio, 95% CI 95% Confidence Interval, ANC Antenatal Care visit, n (%) number of couple counseling events (proportion of couple counseling per modality). Models adjusted on Women age [/table]

Contrast-induced Acute Kidney Injury Contrast-induced acute kidney injury (CI-AKI) is the third common cause of kidney injury in hospitalized patients. It describes a wide spectrum of kidney injury from mild and reversible to permanent and irreversible. The mechanism of contrast-induced AKI and strategies to prevent it are not clearly understood. This review discusses the various contrast agents, pathophysiology of CI-AKI and different preventive strategies. # Introduction Advances in radiodiagnosis and angiography have led to improved clinical diagnosis and safe patient management in recent years. Most modern radiological techniques involve the administration of iodinated radiocontrast material to delineate vascular structures and hollow viscus for accurate diagnosis, and precise interventions. One of the major adverse effects of this iodinated intravascular contrast is contrast-induced acute kidney injury (CI-AKI), formerly known as contrast-induced nephropathy (CIN). Contrast-induced acute kidney injury is the third most common cause for acute kidney injury (AKI) in hospitalized patients. [bib_ref] Hospital-acquired renal insufficiency, Nash [/bib_ref] It is usually mild and reversible, but in some patients, it can cause irreversible kidney injury requiring renal replacement therapy. Since CI-AKI is predominantly related to iodinated contrast, non-iodinated contrast-related renal insufficiency will not be discussed in this review. ## Definition a n d incidence Contrast-induced acute kidney injury is suspected when there is a decline in renal function following recent intravascular administration of contrast media for imaging or interventional purposes. There is no universally accepted definition for CI-AKI. Kidney Disease Improving Global Outcomes guidelines published in 2012 recommended the use of AKI definition for CI-AKI, which is 2 - Absolute increase in creatinine of ≥0.3 mg/dL within 48 hours of administration of intravascular contrast material or - Increase in creatinine of at least 50% above baseline within 7 days of administration of intravascular contrast material or - A urinary volume of less than 0.5 mL/kg of body weight/hour that persists for at least 6 hours after exposure A diagnosis of CI-AKI should be made only after excluding other potential causes of AKI (hemodynamic shock, rhabdomyolysis, use of nephrotoxic drugs and urinary tract obstruction). The incidence of CI-AKI depends on patient factors, type of procedure performed, route of contrast administered and the definition applied.Coronary angiography and angioplasty are more likely to cause CI-AKI than other contrast studies with an incidence estimated between 3% and 13%.In patients with normal renal function, the incidence of CI-AKI is estimated to be between 1% and 2%. [bib_ref] Nephrotoxicity of contrast media, Berns [/bib_ref] In preexisting renal dysfunction with or without other risk factors like advanced age, diabetes, congestive heart failure and administration of other nephrotoxic drugs its incidence can be as high as 25%. [bib_ref] Contrast-induced nephropathy: is the picture any clearer?, Rudnick [/bib_ref] It affects 11% of hospitalized patients and is the third most common cause for AKI in hospitalized patients. [bib_ref] Hospital-acquired renal insufficiency, Nash [/bib_ref] Several small studies have shown an association between CI-AKI and mortality. [bib_ref] Hospital-acquired renal insufficiency, Nash [/bib_ref] [bib_ref] Incidence and prognostic importance of acute renal failure after percutaneous coronary intervention, Rihal [/bib_ref] Majority of these studies included patients undergoing coronary intervention, which makes it impossible to generalize these findings to other patient groups. Moreover, patients with chronic kidney disease (CKD) are increasingly less likely to undergo procedures or imaging that involves the administration of radiocontrast material. Therefore, it is impossible to know whether contrast is truly causative of AKI. Contrast-induced acute kidney injury is likely to prolong hospital stay, but its attributable mortality is still largely unknown. 6 ## Types o f contrast media a n d pathophysiology o f ci-aki Based on their osmolality relative to serum, iodinated contrast media are classified into high, low, and iso-osmolal materials. Highosmolal materials, also called first-generation materials or ionic contrast, typically have 5-8 times the osmolality of body fluids (1400-2200 mOsm/kg water). High incidence of adverse reactions secondary to the osmotic effect led to high-osmolal agents falling out of favor. They are rarely used in clinical practice nowadays. [bib_ref] Intravenous contrast: friend or foe? a review on contrastinduced nephropathy, Do [/bib_ref] Low-osmolal agents (second-generation agents or nonionic contrast) with an osmolality between 600 and 800 mOsm/kg water cause fewer adverse reactions and are well tolerated. Nevertheless, they are still hypertonic and have the potential to cause AKI. They are less expensive and are the most commonly used contrast agents in clinical practice. [bib_ref] Intravenous contrast: friend or foe? a review on contrastinduced nephropathy, Do [/bib_ref] Iso-osmolal contrast agents or third-generation contrast agents have similar osmolality to that of serum. They are well tolerated but have high viscosity and are very expensive. Considering their high cost and the equal to marginally reduced risk of CI-AKI compared to low-osmolal agents, the use of iso-osmolal agents is restricted to patients at high risk of developing CI-AKI. 7 ## Pathophysiology Contrast media can cause kidney injury by direct or indirect mechanisms. 8 ## Indirect mechanisms Contrast agents cause fluctuations in the blood flow both regionally and globally. The outer medulla with its physiologically low oxygen saturation along with high metabolic demand makes it vulnerable for injury when there is associated reduction in blood flow by contrast media. [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] [bib_ref] Intravenous contrast: friend or foe? a review on contrastinduced nephropathy, Do [/bib_ref] The local reduction of blood flow is mediated by vasoactive substances like increased endothelin, renin-angiotensin, and decreased prostaglandin and nitric oxide. With a half-life of around 2 hours, contrast gradually gets excreted through the kidneys. By 24 hours, they are completely excreted from the body. During excretion, they pass through the tubules causing an increased osmotic force which increases sodium and water excretion. This in turn, increases intratubular pressure, thereby reducing the glomerular filtration rate (GFR). Contrast agents also increase blood viscosity, decrease microcirculation, affect the plasticity of erythrocytes and cause microvascular thrombosis. 7 ## Direct mechanisms Iodinated contrast media are toxic to the epithelial cells of the tubules causing necrosis and apoptosis. There is redistribution of basolateral Na + /K + ATPase to luminal surface causing increased Na delivery to the distal tubules. The damaged epithelial cells detach and clog the tubular lumen, increasing intratubular pressure, thereby reducing the GFR further. 8 ## Risk factors f o r ci-aki - Contrast-related factors - Patient-related factors # Contrast-related factors Osmolality, viscosity, volume, and route of contrast administered [fig_ref] Table 1: Contrast media 7 [/fig_ref] are major determinants of CI-AKI. [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] [bib_ref] Intravenous contrast: friend or foe? a review on contrastinduced nephropathy, Do [/bib_ref] Clearly, the risk of developing CI-AKI is much higher with firstgeneration high-osmolal agents. It is widely accepted that high osmolality is associated with AKI, but there is some suggestion that viscosity is more important than osmolality, 7 since it alters the rheologic property of the fluids. Studies comparing lowosmolal and iso-osmolal agents showed no difference in the risk of developing AKI, supporting the viscosity theory. Other important factors are the volume of contrast administered, route of administration 9 and the type of procedure performed. Higher volumes tend to cause more osmotic effect, reduce GFR, stay in the tubules for longer and cause direct injury to the tubular cells. [bib_ref] Contrast-induced acute kidney injury, Mccullough [/bib_ref] Incidence of CI-AKI is more after intra-arterial administration of contrast than intravenous administration. Angiography (arterial or venous) certainly carries a much higher risk compared with contrast-enhanced computed tomography. 7 # Patient-related factors Incidence of CI-AKI increases with age, preexisting renal disease, diabetic nephropathy, simultaneous use of nephrotoxic drugs, dehydration, hemodynamic instability, anemia and other causes of reduced renal perfusion. [bib_ref] Incidence and prognostic importance of acute renal failure after percutaneous coronary intervention, Rihal [/bib_ref] [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] [bib_ref] Low hematocrit predicts contrast-induced nephropathy after percutaneous coronary interventions, Nikolsky [/bib_ref] [bib_ref] Radiocontrast nephropathy: a paradigm for the synergism between toxic and hypoxic insults..., Lang [/bib_ref] Since these factors are associated with low renal reserve, administration of contrast increases the risk of developing AKI. Several prediction models have been proposed to identify patients at risk of developing CI-AKI. [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] These models have incorporated various patient, procedural, and contrast-related factors to arrive at a final score to predict the risk. [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] They were constructed using large cohorts of patients undergoing coronary interventions. One such model proposed by Mehran et al. [bib_ref] A simple risk score for prediction of contrast-induced nephropathy after percutaneous coronary..., Mehran [/bib_ref] is given in [fig_ref] Table 2: Mehran et al [/fig_ref]. Although not widely used, they are quite useful in predicting AKI in patients undergoing coronary intervention. ## Recent developments i n prediction a n d diagnosis Prediction and diagnosis of AKI from contrast administration involves a careful assessment of patient-related risk factors, volume of contrast used, interval between contrast administration, and the change in renal parameters. [bib_ref] Contrast-induced nephropathy: basic concepts, pathophysiological implications and prevention strategies, Mamoulakis [/bib_ref] Changes in the fluid balance have an effect on serum creatinine which affects the diagnosis of AKI. In some studies, paradoxically, a decreasing serum creatinine was associated with increased mortality in postsurgical patients. [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] This uncertainty with creatinine led to the search for biomarkers, and bedside ultrasound has been suggested to predict and diagnose CI-AKI. ## S124 ## Biomarkers These can be used to detect AKI post-contrast exposure. Although helpful in early detection of AKI before a change in creatinine, these biomarkers are not specific for CI-AKI. Biomarkers like urinary neurogelatinase-associated lipoprotein [bib_ref] Neutrophil gelatinase-associated lipocalin in the prediction of contrast-induced nephropathy, Tong [/bib_ref] and urinary interleukin-18 indicate renal injury within 2-4 hours of insult. Other biomarkers like fatty acid-binding protein and kidney injury molecule-1 also indicate toxic injury to the renal tubules. ## Renal-resistive index 15 Assessing renal-resistive index (RRI) with bedside ultrasound to predict AKI has been studied lately. Like the biomarkers, it is not specific for CI-AKI. The changes in renal blood flow detected by RRI help detect various renal pathologies. It has been used to assess post-renal transplant allograft function, renovascular hypertension, etc. A high RRI before administration of contrast is a good predictor of CI-AKI. ## Treatment There is no specific treatment for CI-AKI. The treatment is similar to AKI from other causes. Hence, strategies have been the suggested and followed to prevent the development of CI-AKI. ## Preventive strategies: nonp h a r m aco lo g i c a l Identifying patients at risk and weighing the risks and benefits of administering contrast with a view to reducing the risk prevent the morbidity associated with CI-AKI. If the risks far outweigh the benefits, it becomes important to explore alternate imaging modalities to avoid contrast exposure. Well-established preventive strategies are as follows: - Using the minimum volume of contrast possible without compromising the image quality. [bib_ref] Does safe dosing of iodinated contrast prevent contrastinduced acute kidney injur y?, Brown [/bib_ref] - Avoiding repeat exposure to contrast within 72 hours. [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] - Assessing and adequately addressing volume depletion, thereby avoiding activation of renal angiotensin mechanism which leads to vasoconstriction. Improved urine flow in the tubules prevents contrast stasis and hence reduces the direct injury risk. 17 - Avoiding concurrent usage of nephrotoxic drugs. 17 ## Volume of contrast media These that can be used safely with minimal effects on kidney have been widely studied. Brown et al. [bib_ref] Does safe dosing of iodinated contrast prevent contrastinduced acute kidney injur y?, Brown [/bib_ref] in their study introduced maximum acceptable contrast dose which is calculated as 5 × body weight/88.4 × serum creatinine (mmol/L). Dose exceeding this has been associated with AKI. Repeated administration within 72 hours is also associated with higher risk. ## Concurrent use of nephrotoxic drugs Angiotensin-converting enzyme (ACE) inhibitors, nonsteroidal anti-inflammatory drugs (NSAIDs), cyclooxygenase-2 inhibitors, metformin and antivirals like acyclovir and foscarnet increase the risk of AKI in patients exposed to contrast materials. In practice, for elective cases, ACE inhibitors are discontinued on the day of contrast administration, but this has not been proven to be beneficial. Avoidance of NSAIDs is appropriate. Metformin is generally held a day before contrast exposure and has been shown to be beneficial. It is not nephrotoxic, but it causes lactic acidosis if the patient were to develop AKI post-contrast. 6 ## Remote ischemic preconditioning It is believed that a transient hypoperfusion or ischemia in one organ/area of the body has protective effects in others. Intermittent inflation of an upper arm cuff arm causing ischemia reduces the risk of CI-AKI in high-risk patients. [bib_ref] Remote ischemic preconditioning alleviates contrast induced acute kidney injury in patients with..., Igarashi [/bib_ref] Well-validated clinical studies are needed before this becomes a regular practice. ## Preventive strategies: pharmacological Volume expansion with normal saline is found to be the most physiologically appropriate strategy to reduce the risk of CI-AKI. Saline improves the urine flow hence dilutes the contrast media and reduces its concentration and osmolality. This reduces the intraluminal block and back pressure. Volume expansion also inhibits the renin-angiotensin system, thereby preventing renal vasoconstriction. [bib_ref] Effectiveness of prevention strategies for contrastinduced nephropathy, Subramaniam [/bib_ref] [bib_ref] The prevention and management of contrast-induced acute kidney injury: a mini-review of..., Ali [/bib_ref] [bib_ref] What is the best hydration regimen to prevent contrast mediainduced nephrotoxicity?, Bader [/bib_ref] Although observational data to support volume expansion is very strong, clinical trials have thus far failed to favor this preventive strategy. This could be due to various factors including methodological issues. The volume of intravenous saline required for prevention is still unknown and is likely to be different for different patients. Despite this lack of clarity, American College of Radiology recommends 100 mL/hour saline for 6-12 hours before and 4-12 hours after any angiography. Since this is not practical for urgent procedures, intravenous fluids can be given for 1-3 hours before and 6 hours after an urgent procedure. Patients with poor left ventricular function and chronic kidney disease are at risk of overload; therefore, volume expansion needs to be done cautiously. [bib_ref] Contrast-associated acute kidney injury, Mehran [/bib_ref] [bib_ref] The prevention and management of contrast-induced acute kidney injury: a mini-review of..., Ali [/bib_ref] [bib_ref] What is the best hydration regimen to prevent contrast mediainduced nephrotoxicity?, Bader [/bib_ref] Sodium bicarbonate (NaHCO 3 ) 17 is thought to reduce free radicals and increase neutralization of active oxygen species produced by contrast in renal tubules by alkalinizing the urine. A large multicenter randomized controlled trial comparing saline and bicarbonate did not show any difference between the two interventions. Therefore, routine use of bicarbonate infusion to prevent CI-AKI cannot be recommended. Antioxidants such as N-acetyl cysteine 17 are expected to be effective due to their ability to reduce/counteract the free radicals produced in the tubule. N-Acetyl cysteine is commonly used in clinical practice to reduce the risk of CI-AKI. Although retrospective clinical data support its usage, large clinical trials have not shown any benefit thus far. Its routine use to prevent AKI is not recommended. Theophylline 17 is a nonselective adenosine receptor antagonist. Adenosine is believed to mediate vasoconstriction after contrast administration. This mechanism is found to be negated by theophylline. Although theoretical explanation holds good, studies did not show a beneficial role with the use of theophylline. Rather this drug had significant gastrointestinal and cardiovascular side effects. Statins 17 (3-hydroxy-3-methyl-glutaryl-CoA reductase inhibitors) are also known to be anti-inflammatory and have antioxidant properties. Multiple studies have evaluated the effect of short-term high-dose statins in preventing CI-AKI. Despite the proposed benefits, they are not widely used because of differing study protocols and conflicting results of the trials. Other drugs such as calcium channel blockers, allopurinol, fenoldopam, dopamine and prostaglandin E1 have all been studied to prevent CI-AKI, but the lack of evidence does not support the use of these agents in clinical practice. [bib_ref] Effectiveness of prevention strategies for contrastinduced nephropathy, Subramaniam [/bib_ref] newer Methods o f non-contrAst IMAgIng 21 Newer imaging techniques without the need for iodinated contrast are being considered recently. These are mostly magnetic resonance-based studies. They are not widely available, and many are specialized studies which require skill and expertise [fig_ref] Table 3: Imaging techniques without contrast usage 21 TOF, time-of-flight [/fig_ref]. # Conclusion Despite the many unknowns surrounding CI-AKI, it must be acknowledged that it is a recognized problem in patients undergoing contrast-enhanced studies and procedures. Judicious use of contrast after a careful risk assessment and application of appropriate preventive measures are likely to reduce the risk of developing CI-AKI. ## References [table] Table 1: Contrast media 7 [/table] [table] Table 2: Mehran et al. 's risk sore for contrast-induced nephropathy6,12 [/table] [table] Table 3: Imaging techniques without contrast usage 21 TOF, time-of-flight; MR, magnetic resonance, ECG, electrocardiography, SSFP, steady-state free precession; AV, arteriovenous [/table]

Costs of remote monitoring vs. ambulatory follow-ups of implanted cardioverter defibrillators in the randomized ECOST study AimsThe Effectiveness and Cost of ICD follow-up Schedule with Telecardiology (ECOST) trial evaluated prospectively the economic impact of long-term remote monitoring (RM) of implantable cardioverter defibrillators (ICDs).Methods and resultsThe analysis included 310 patients randomly assigned to RM (active group) vs. ambulatory follow-ups (control group). Patients in the active group were seen once a year unless the system reported an event mandating an ambulatory visit, while patients in the control group were seen in the ambulatory department every 6 months. The costs of each follow-up strategy were compared, using the actual billing documents issued by the French health insurance system, including costs of (i) (a) ICD-related ambulatory visits and transportation, (b) other ambulatory visits, (c) cardiovascular treatments and procedures, and (ii) hospitalizations for the management of cardiovascular events. The ICD and RM system costs were calculated on the basis of the device remaining longevity at the end of the study. The characteristics of the study groups were similar. Over a follow-up of 27 months, the mean non-hospital costs per patient-year were E1695 + 1131 in the active, vs. E1952 + 1023 in the control group (P ¼ 0.04), a E257 difference mainly due to device management. The hospitalization costs per patient-year were E2829 + 6382 and E3549 + 9714 in the active and control groups, respectively (P ¼ 0.46). Adding the ICD to the non-hospital costs, the savings were E494 (P ¼ 0.005) or, when the monitoring system was included, E315 (P ¼ 0.05) per patient-year.ConclusionFrom the French health insurance perspective, the remote management of ICD patients is cost saving.Clinical trials registrationNCT00989417, www.clinicaltrials.gov --- # Introduction Remote monitoring (RM) of implantable cardioverter defibrillators (ICDs) was introduced just over10 years ago and its use is rapidly increasing. [bib_ref] ISHNE/EHRA expert consensus on remote monitoring of cardiovascular implantable electronic devices (CIEDs), Dubner [/bib_ref] The clinical relevance of remotely monitoring ICD recipients has been confirmed. Randomized studies have shown that RM was as safe as conventional ambulatory follow-ups. [bib_ref] ECOST trial Investigators. A randomized study of remote follow-up of implantable cardioverter..., Guédon-Moreau [/bib_ref] [bib_ref] Efficacy and safety of automatic remote monitoring for implantable cardioverterdefibrillator follow-up: The..., Varma [/bib_ref] Most importantly, RM was highly effective in lowering the incidence of appropriate and inappropriate shocks. [bib_ref] ECOST trial Investigators. A randomized study of remote follow-up of implantable cardioverter..., Guédon-Moreau [/bib_ref] Furthermore, it shortens the medical follow-ups of ICD recipients. [bib_ref] Assessment of a remote monitoring system for implantable cardioverter defibrillators, Masella [/bib_ref] [bib_ref] Workload and usefulness of daily, centralized home monitoring for patients treated with..., Vogtmann [/bib_ref] Besides these obvious benefits, its economic value needed to be examined, [bib_ref] Remote monitoring: a cost or an investment?, Burri [/bib_ref] as several studies suggested that RM might be cost saving compared with ambulatory follow-ups. However, these studies used expected costs based on estimates and did not take into account the multiplicity of variables that determine costs. The ECOST trial was designed to compare prospectively the safety and the costs of remote ICD monitoring with standard ambulatory follow-ups. The safety results have been previously reported. [bib_ref] ECOST trial Investigators. A randomized study of remote follow-up of implantable cardioverter..., Guédon-Moreau [/bib_ref] We present here the results of the economic analysis. # Methods ## Trial design We randomly assigned patients, in a 1 : 1 design, to RM (active group) vs. ambulatory follow-ups (control group), before they underwent implantation of single-or dual-chamber ICD, and after they had granted written consent to participate in the study. The protocol of ECOST has been previously published. [bib_ref] ECOST trial Investigators. A randomized study of remote follow-up of implantable cardioverter..., Guédon-Moreau [/bib_ref] The study included 433 recipients of commercially available ICD equipped with Biotronik Home Monitoring w (HM; Biotronik SE and Co. KG), a wireless communication system, which automatically transmits diagnostic data and trend analyses between the implanted device and the caregiver on a daily basis. The system and its operating mode have been previously described. [bib_ref] Efficacy and safety of automatic remote monitoring for implantable cardioverterdefibrillator follow-up: The..., Varma [/bib_ref] [bib_ref] Remote, wireless, ambulatory monitoring of implantable pacemakers, cardioverter defibrillators, and cardiac resynchronization..., Lazarus [/bib_ref] [bib_ref] ECOST trial Investigators. Contributions of remote monitoring to the follow-up of implantable..., Guédon-Moreau [/bib_ref] Patients assigned to the active group were followed with HM and were seen by a cardiac electrophysiologist in the ambulatory department within 1 -3 months of ICD implantation, and at Months 15 and 27 of follow-up, thereafter. At any time, additional ambulatory visits could be triggered by HM data or scheduled at the request of the patient or a physician. In the control group, the patients were seen in the ambulatory department at 1 -3 months after ICD implantation for a first follow-up, and at 9, 15, 21, and 27 months of follow-up, thereafter. Additional visits could be scheduled if requested by the patient or a physician. In addition, all study participants were followed by a cardiologist, as needed, for management of their underlying heart disease. The trial protocol, which complied with the declaration of Helsinki, was reviewed and approved by the pertinent ethics committees. All patient information and data collected were treated confidentially by the sponsor and all other parties involved in the trial, and all clinical centres were regularly monitored by the study team. # Costs analysis The costs, presented per patient per year, were calculated in Euros (E), using the actual charges entered in the database of the French national health insurance billing system. Their written authorization was requested to the patients to anonymously retrieve pertinent data from the system, based on social security numbers, including (i) direct costs of ambulatory ICD follow-ups and associated transportation expenses, (ii) direct costs of ICD-unrelated ambulatory follow-ups, cardiovascular treatments and procedures, and (iii) direct hospital costs for management of cardiovascular disorders. The classifications used by the health insurance databases, such as diagnosis-related groups, major diagnostic categories and the classification of medical acts or drug treatments ,http://www.ameli.fr., were used to differentiate and analyse cardiovascular hospitalizations, cardiovascular procedures, and drugs costs separately. The hospitalization cost was determined by the health insurance system by its diagnosis-related group class, which is weighted by the hospitalization duration and associated co-morbidities. The approximately E60 medical charges for ambulatory follow-up of ICD and transportation costs were specifically identified in the databases by the dates of follow-ups collected during the study. The travel reimbursements were based on a flat rate, mainly related to the (i) types of transportation, such as ambulance, taxi, personal vehicle or public transportation, and (ii) mileage. A cost analysis compared the individual costs in each study group, during the study period. Incurred and reimbursed costs per patient-year were analysed, as well as their difference, reflecting the costs supported by the patient or by a supplemental insurance. The French national health insurance system typically covers 55 -75% of non-hospital costs, 80% of the hospital costs, and 65% of the transportation costs, though chronically ill patients are 100% covered. In addition, co pays are collected from the patients for each reimbursed service, including E18 for hospitalizations, E1 for consultations, E2 for transportation, and E0.5 for drug prescriptions. Incurred, reimbursed and supported costs used for this study were all recorded in the national health insurance databases, except for the additional costs generated by the management of the RM which are not yet reimbursed in France and have therefore not been accounted for the analysis. The costs of ICD are invoiced in addition to the diagnosis-related groups and also covered by the health insurance system. Their prices, which are set by a France's economics committee for health products, appear on the list of products and services. At the time of this study, the prices and reimbursements were E12 000 for a single-chamber and E14 000 for a dual-chamber ICD. The cost of ICD per patient was calculated as a function of the remaining device longevity at the end of the study, which was evaluated by the slope of battery depletion over time, using a linear regression model. In case of death or device explantation before the end of the study, the full price of a partially unused device was entered in the analysis. The costs of the hardware and service provided for the telemonitoring were not taken into account in the analysis since, as they were not covered by health insurance at the time of the study, they were not invoiced by the manufacturer. # Patient acceptance analysis Additional variables that we analysed included patient quality of life and willingness to pay for HM services. The quality of life was measured at several stages of the study, including (i) enrolment, (ii) first follow-up, (iii) 15-month follow-up, and (iv) last follow-up, using the SF-36 questionnaire. The benefits conferred to the patients were measured as willingness to pay, i.e. the monetary amount which they were willing to pay for the HM service, as well as the preference expressed for one vs. the other type of follow-up. This was ascertained at the end of the study by a questionnaire probing into (i) preferred ambulatory follow-ups every 6 months, (ii) preferred HM + yearly ambulatory follow-ups, or (iii) no preference. # Statistical analysis After confirmation of the statistical non-inferiority of the primary safety endpoint of the study, by comparing the proportion of patients who What's new? † Effectiveness and Cost of ICD follow-up Schedule with Telecardiology is the first study that has evaluated the real economic impact of long-term remote monitoring (RM) of implantable cardioverter defibrillators (ICDs), using the actual billing documents issued by the French health insurance system. † After it was shown to be safe and clinically efficacious, ICD RM was found cost-saving. Device management with RM optimizes patient care and clinical resources, without supplementary costs. † The findings on this study have direct implications on the management of ICD patients and may therefore impact clinical practice. Remote monitoring tends to become the new gold standard of care for ICD recipients follow-up. experienced one or more major adverse event in each group, 2 we analysed the costs of healthcare associated with each follow-up strategy. Consecutive costs incurred and reimbursed in both study groups throughout the entire study period were calculated per patient-year and reported as means, standard deviations, medians and inter-quartile ranges. Because the data were not normally distributed, a non-parametric bootstrap method was used to derive bias-corrected confidence levels (10 000 replications). [bib_ref] Analysis of cost data in randomized trials: an application of the non-parametric..., Barber [/bib_ref] The mean between-group differences were compared, using Student's t-test with bootstrap-based P values. The baseline nominal characteristics of the study groups were compared by x 2 test. The normal distribution of variables was verified, using the Kolmogorov -Smirnov and Shapiro-Wilk tests. Normally distributed variables were compared, using Student's t-test, after confirmation of the equality of variances by Levene's test. Mann-Whitney's non-parametric test was used to compare the SF-36 scale data and summary scores between both the groups. All tests were performed at a P value of 0.05 significance level. The SPSS, version 18.0 (SPSS Institute, Inc.) and R, version 2.14.1 statistical software, were used for the analyses. # Results Among the 433 study participants, 310 (71.6%) granted permission to access their health insurance data and comprised the study population for this economic analysis, with clinical characteristics similar to the initial population. The baseline clinical characteristics of the158 patients in the active and 152 patients in the control group were also similar [fig_ref] Table 1: Baseline characteristics of patients included in the economic analysis of the ECOST... [/fig_ref]. The mean follow-up duration was 26.5 + 3.4 months. ## Direct, non-hospital costs reimbursed by healthcare insurance The costs of non-hospital care, including (i) costs related to the device management and (ii) other non-hospital costs, were 13% lower in the active than in the control group, corresponding to a E257 (95% CI: 5-489) cost saving per patient-year (P ¼ 0.04), a decrease attributable particularly to the lower costs of device management. ## Costs related to the device management The mean costs of non-hospital care for device management, including ICD-related ambulatory visits and associated transportation costs, were 26% lower in the active than in the control group, representing a mean E74 (95% CI: 30-118) saving per patient-year (P ¼ 0.001,. The cost saving per patient/year was E50 (95% CI: E4 -96), an 18% reduction (P ¼ 0.04), for the first 15 months, and E110 (95% CI: E62-160), a 36% reduction (P , 0.001), for the last 12 months of follow-up. ## Costs of ambulatory visits for icd management The costs of ambulatory visits for ICD management were dependent on the number of follow-ups in each group. During the 27-month follow-up period, the mean number of scheduled or additional ambulatory follow-ups per patient-year was 26% lower (P , 0.001) in the active than in the control group. The costs of these followups were 29% lower in the active than in the control group, representing a mean E40 cost saving per patient-year. ## Transportation costs The transportation costs hinged on (i) the number of ambulatory follow-up visits, (ii) the proportion of patients whose transportation costs were reimbursed, (iii) the transportation distance, and (iv) the type of transportation. In both the groups, 48% of patients had reimbursed transportation costs, though this 17% between-groups difference was not statistically significant (P ¼ 0.11). Other direct, non-hospital costs reimbursed by the healthcare insurance The other non-hospital costs were similar in both groups. ## Direct hospital costs reimbursed by healthcare insurance No significant between-groups difference was observed in the costs of hospitalizations for management of cardiovascular disorders. ## Costs supported by the patients The costs supported by the patients for non-hospital-based device management [fig_ref] Figure 1: Costs incurred, reimbursed by the healthcare insurance and supported by the patients,... [/fig_ref] , including (i) non-reimbursed transportation costsValues are means + SD and median with inter-quartile in euros (E); bootstrap results of 10 000 samples (bias corrected) with replacement for the difference in mean cost comparisons between both groups; CI, confidence interval. ## Costs of devices The mean estimated ICD costs per patient, based on the remaining longevity of the devices after the 27 months of follow-up, were E5832 + 2047 in the active vs. E6365 + 3125 in the control group (bootstrap-based P ¼ 0.08). ## Quality-of-life estimates The between-and within-groups differences in mean physical, psychological, and overall SF-36 quality-of-life scores were all statistically non-significant. ## Patient willingness to pay The patient preferred ICD follow-up strategy was based on 194 (44.8%) questionnaires completed at the end of the study. A preference in favour of the active group follow-up strategy was expressed by 73.7% of patients assigned to the active group vs. 65.3% assigned to the control group (P ¼ 0.21), while 7.1% of patients in the active and 14.7% in the control groups expressed a preference in favour of the follow-up strategy applied in the control group. The remaining patients expressed no preference. The mean amount that the patients were willing to pay for the use of HM was E84 + 112 per month (median E43; P ¼ 0.11 between both groups). # Discussion In this randomized comparison, we found that, over a follow-up of 27 months after ICD implantation, the direct non-hospital-related costs of HM were 26% lower than the costs of ambulatory follow-up. This decrease was not associated with other higher costs, such as those incurred for ambulatory visits unrelated to the ICD, or for cardiovascular procedures or hospitalizations for the management of cardiovascular disorders. While the methodology of medico-economical studies is often criticized, [bib_ref] Remote monitoring: a cost or an investment?, Burri [/bib_ref] [bib_ref] Systematic review of studies of the cost-effectiveness of telemedicine and telecare. Changes..., Mistry [/bib_ref] Effectiveness and Cost of ICD follow-up Schedule with Telecardiology was based on a rigorous methodology, which yielded reliable results. The strengths of ECOST are (i) its randomized and controlled design, (ii) the clear definition and homogeneity of the study sample, (iii) the reliability and comprehensiveness of the sources of cost information, (iv) its duration, and (v) the combined cost analysis and evaluation of the efficacy of the follow-up strategies. These strengths separate ECOST from earlier publications pertaining to the RM of ICD recipients. [bib_ref] Potential cost savings by telemedicine-assisted long-term care of implantable cardioverter defibrillator recipients, Fauchier [/bib_ref] [bib_ref] Remote monitoring of implantable cardioverter defibrillator patients: a safe, time-saving, and cost-effective..., Raatikainen [/bib_ref] It was worthwhile to focus the cost analysis on the direct nonhospital costs and the costs related to the devices, both of which might be influenced by the RM strategy. While one might suspect the RM to shorten the life of the ICD battery because of the energy consumed by data transmission, it is quite the opposite. This slight consumption was more than compensated by the battery saving due to fewer delivered or undelivered capacitor charges during the study. [bib_ref] ECOST trial Investigators. A randomized study of remote follow-up of implantable cardioverter..., Guédon-Moreau [/bib_ref] In those two cost items, the saving attributable to RM was E494 per patient per year. This saving represents Cost of remote ICD follow-up $430 per patient per year, including $121 per transportation and $94 per medical service. [bib_ref] Potential cost savings by telemedicine-assisted long-term care of implantable cardioverter defibrillator recipients, Fauchier [/bib_ref] The savings recorded in ECOST were lower because the study protocol had planned a difference of a single annual visit between the two study groups, and because the mean costs for medical services were actually lower. Effectiveness and Cost of ICD follow-up Schedule with Telecardiology also accounted for additional ICD consultations prompted by HM notification events or requested by patients or physicians. Furthermore, in the study by Fauchier et al., the costs covered by third parties were not distinguished from those supported by the patients, whereas only 48% of patient transportations costs in ECOST were absorbed by the health insurance. While one might have expected a parallel increase in costs other than those directly related to the ICD, for example a greater number of supplemental general cardiology visits counterbalancing the decrease in the number of ICD-related ambulatory visits, this was not the case. The savings observed in this study were particularly significant in view of the greater efficacy of RM (fewer shocks delivered) combined with its equivalent safety (no increase in major adverse events) compared with ambulatory visits. 2 Moreover, the large epidemiological ALTITUDE study, which included nearly 186 000 patients, suggested that RM could have a positive impact on survival, even though this result has to be confirmed by a controlled study. [bib_ref] Long-term outcome after ICD and CRT implantation and influence of remote device..., Saxon [/bib_ref] From the patient standpoint, as expected, RM did not significantly lower the direct costs, since the health insurance system in France covers nearly all expenses, particularly in the case of chronic illnesses. Indirect costs, such as lost wages, were not included in this analysis, because they were not important enough, as a large proportion of patients were retired or on disability. Neither did we include a cost-utility analysis based on the quality-of-life questionnaire, as it seemed overly generic and insufficiently specific. The overall costbenefit analysis, however, was clearly in favor of RM. From the perspective of healthcare providers, the ambulatory follow-ups are highly time-consuming, with a mean 27 min for scheduled visits measured in the joint EHRA-Eucomed survey. [bib_ref] European Heart Rhythm Association; Eucomed. Healthcare personnel resource burden related to in-clinic..., Boriani [/bib_ref] Remote monitoring, on the other hand, is time saving. [bib_ref] Assessment of a remote monitoring system for implantable cardioverter defibrillators, Masella [/bib_ref] [bib_ref] Workload and usefulness of daily, centralized home monitoring for patients treated with..., Vogtmann [/bib_ref] [bib_ref] Economic impact of remote monitoring on ordinary follow-up of implantable cardioverter defibrillators..., Calo [/bib_ref] The equipment of patients with transmitters and their registration on the site of telemonitoring can be assumed by paramedical staff. Furthermore, this time is more than compensated by the decreased number of ambulatory visits. An analysis of the REFORM trial found that RM saved 81 h of medical time per year per 100 patients followed. [bib_ref] A prospective multicenter comparison trial of home monitoring against regular follow-up in..., Elsner [/bib_ref] The HM system is highly automatized, and the review of ICD data posted on the Internet site is useful only after an event notification of an adverse rhythm or technical event. In our experience, the yearly number of notifications per patient for single-or dual-chamber ICD is 3.5, and the time spent by physicians and nurses in the management of event notifications is 7 and 9 min per patient per year, respectively. This workload generates a cost that only slightly minimizes the cost saving. This performance level is achievable only with an appropriate alert system, which no longer depends on audible or tactile warnings, which disturb the patient with variable degrees of relevancy. Finally, from the hospital perspective, one may surmise that the cost of hospitalizations is lowered by RM because of a shorter mean duration of hospitalization. [bib_ref] The CONNECT (Clinical Evaluation of Remote Notification to Reduce Time to Clinical..., Crossley [/bib_ref] Limitations of the study Effectiveness and Cost of ICD follow-up Schedule with Telecardiology evaluated a single RM system, Biotronik Home Monitoring w . One might hypothesize that other systems, which operate with different functions, might not be associated with the same cost differences, compared with ambulatory follow-ups. The costs of the system itself were not included in this analysis because, in France, these costs were absorbed by the device manufacturer at the time of the study. Since 2011, however, the HM system is reimbursed by the French healthcare insurance, at a rate of E1000, which includes the transmission costs and the service for each implanted device for its entire life. If this cost, based on the remaining device longevity at the end of the study, is added to the direct non-hospital costs and the costs related to the devices, the cost saving is lowered by only 7% (E4466 + 1492 per patient-year in the active vs. E4781 + 1594 per patient-year in the control group) and remains a substantive E315 per patient-year. Furthermore, although the medical staff used additional time to manage event notifications in the group followed remotely, our analysis did not include a pricing for remote follow-up, a reimbursement that does not currently exist in France. In view of cost savings of E315 per patient-year highlighted in ECOST and according to the reimbursement that could be implemented for the medical staff, a cost balance between both monitoring strategies could be obtained. In addition, the field of healthcare financing is complex and financing modalities differ greatly among countries. [bib_ref] Device therapy and hospital reimbursement practices across European countries: a heterogeneous scenario, Boriani [/bib_ref] The results of the ECOST apply to the French system. However, the details of the study methodology which are provided should allow a conversion of the results to different funding systems. Finally, our results were obtained in recipients of ICD without resynchronization therapy. Compared with our sample, cardiac resynchronization therapy defibrillator (CRT-D) recipients typically suffer from more severe heart disease and generate higher medical costs. However, in a previous study, RM reduced the total amount of healthcare consumption, including by patients suffering from heart failure who were recipients of ICD or CRT-D. [bib_ref] Remote monitoring reduces healthcare use and improves quality of care in heart..., Landolina [/bib_ref] # Conclusion After it was shown to be safe and clinically efficacious, RM of ICD was found cost saving from the French health insurance perspective. Remote management of ICD patients meets the criteria proposed by Scott for the validation of activities of telemedicine, which are quality, accessibility, cost and acceptance criteria. [bib_ref] Telehealth outcomes: a synthesis of the literature and recommendations for outcome indicators, Scott [/bib_ref] ## Supplementary data The French investigators and institutions that participated in the ECOST trial are given as supplementary data available at Europace online. ## Conflict [fig] Figure 1: Costs incurred, reimbursed by the healthcare insurance and supported by the patients, per patient-year. The costs supported by the patients are the difference between the costs incurred and the amount reimbursed by the health insurance. (A) The costs related to the device management, including ambulatory follow-ups of ICD and related transportations. (B) The addition of the device management costs and other nonhospital costs. (C) Costs of hospital care for the management of cardiovascular disorders. (D) Addition of non-hospital and hospital costs. The P values correspond to the between-group differences in incurred costs. The P values corresponding to differences in costs supported by patients are shown in the text. [/fig] [table] Table 1: Baseline characteristics of patients included in the economic analysis of the ECOST study [/table] [table] Table 2: Mean hospital and non-hospital costs per patient-year reimbursed by healthcare insurance in each study group [/table] [table] Table 3: Transportation costs for ICD ambulatory visits, reimbursed by insurance or supported by patients [/table]

Peptide mimetics of immunoglobulin A (IgA) and FcαRI block IgA‐induced human neutrophil activation and migration [fig] Supporting, Figure 1 Supporting, Figure 3: Model of interaction sites of IgA and FcaRI (A) Model of the structure of the complex of two IgA Fc heavy chains, interacting with two extracellular domains of FcαRI. (Two molecules of FcαRI form a complex with one IgA molecule). (B and C) Magnification of position of FcαRI sequences (B) and IgA sequences (C) involved in binding. Peptide mimetics do not block IL-8 induced chemotaxis Number of fluorescently labelled neutrophils which migrated to IL-8 in a chemotaxis chamber, either in the presence or absence of peptide mimetics. The number of migrated neutrophils was determined with a fluorimeter. Neutrophil migration to IL-8 is indicated with a dotted line. Medium and blocking IL-8 antibody (anti-IL-8) were used as controls. Experiments were performed three times in triplo. Mean ± SD of one representative experiments in triplo is shown. [/fig] [fig] Figure 2: Oxidated or small peptide mimetics do not block IgA-induced migration in vitroPercentage of migration of fluorescently labelled neutrophil to IgA-coated beads, either in the presence or absence of peptide mimetics. The number of migrated neutrophils was determined with a fluorimeter. Neutrophil migration to IgA was normalized to 100% (dotted line). Neutrophils or beads were pre-incubated with (A) oxidated or (B) small cyclic peptides mimicking FcαRIsequences (white bars) or IgA-sequences (black bars). Data are representative of 3 independent experiments, performed in triplicates. Mean ± SD of one representative experiment is shown. Statistical analysis: ANOVA [/fig]

An Efficient, Counter-Selection-Based Method for Prophage Curing in Pseudomonas aeruginosa Strains Inducible phage extraction LB (2 ml) was inoculated with 39016 strain incubated overnight. On the next day, the bacteria were diluted 1:50 with medium to a final volume of 4 ml and incubated until reaching OD 0.5 at 595nm (~2 hours). For phage induction, 0.4 μg/ml norfloxacin (sigma) antibiotic was added, and the cultures were incubated for 1 hour. Fresh LB was added (1.75 mL), and the cultures were incubated for an additional one hour. Then, 1 ml of bacteria was centrifuged at 14,000g for 2 min, and 900 µl of the supernatant was filtered using a 0.45 µm filter (Whatman). For PCR amplification, the extracted phages were treated with 1 μg/ml DNaseI for 60 min at 37°, followed by inactivation of the enzyme activity by incubation at 65° for 15 min.Growth curve LB (2 mL) was inoculated with bacterial strains from frozen stocks and incubated overnight at 37°C with shaking (250 rpm). The culture was diluted to 0.005 OD (595 nm) in fresh media and transferred to a 96-well plate, 200μl in each well. The plates were incubated for 15 hours at 370C with agitation. Optical density measurements at 595 nm were taken every 30 minutes using the Synergy™ 2 Multi-Detection Microplate Reader (BioTek).Primer Sequence M13_F CCCAGTCACGACGTTGTAAAACG M13_R AGCGGATAACAATTTCACACAGG AmpR_F CGCGGAACCCCTATTTGTT AmpR_R TTACCAATGCTTAATCAGTGAGG PR2_500Dn_R AAGCTTAGGCGATCCAGGCCGAC PR2_500up_F GAGCTCTATTTTTATTGCGACGACAGCG PR2_AmpRin _Seq_F GAGCTCATAGATCACCCCCTTGCTCG PR2_AmpRin_Up_F_GWB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTCACAAGCCATTTGCACCAGCAG PR2_AmpRin_Up_R AACAAATAGGGGTTCCGCGGATACCATGAAACGAGCAACC PR2_AmpRin_Down_F CCTCACTGATTAAGCATTGGTAACTACTTGCTGATGCCGATGAA PR2_AmpRin_Down_R_GWB2 GGGGACCACTTTGTACAAGAAAGCTGGGTAGTGACGACGCTGTTCCAGA PR2_AmpRin _Seq_R GAATTCCGTTGAGGGTATGAAGGTTG PR2_SacBin_Up_F_GWB1 GGGGACAAGTTTGTACAAAAAAGCAGGCTCAAACACGGCGAAGCAGAGCT PR2_SacBin_Up_R GCGCGCACGTATCAACAGATTCAAGCCGCATCGAGCAAC PR2_ SacBin _Down_F ATCTGTTGATACGTGCGCGC PR2_SacBin_Down_R_GWB2 GGGGACCACTTTGTACAAGAAAGCTGGGTATAGCTGCGTGTTGGCTTGC Pf4_RF_F AGCAGCGCGATGAAGCAAT Pf4_RF_R TAGAGGCCATTTGTGACTGGA Pf4_seq_F TACGAGGCTGTTGAGGAGTTA Pf4_seq_R CCGTGCCGAGGTAGTGATGTC PR5_seq_F TCAAACCATCCAATAGCTGGC PR5_seq_R GGCGGCAGGCGTATCCTT PA39016_100004_F ATGGTTAAGAAATTCTCCGAC PA39016_100004_R TTAGGCGCCCGCCTCTTC : ampR insertion into the PR2 prophage did not alter the bacterial growth and PAO1infectious phages production. (A) The growth curve of ampR inserted strain (AmpRin) compared to WT 39016; OD was measured automatically every 30 min. (B) Plaque forming units count, PAO1 was used as a host, phages were induced and extracted from WT, and the ampR inserted strain (AmpRin). The above graphs are the average of two independent experiments consisting of (A) five replicates each (B) three replicates each. Error bars represent the standard deviations. [fig] Figure S2: The SacBin region presents in PR2-cured colonies. PCR amplification of 1100bp SacBin region; lanes 1-8 represent randomly picked Crb-sensitive colonies, lanes 9-10 represent Crb-resistant colonies, and the WT 39016 lane for positive control. The PR2_SacBin_Up_F_GWB1 and SacBin_Down_R_GWB2 were used for the amplification. [/fig] [fig] Figure S3: PR2 phages are not produced in the cured strain. PCR amplification of 1200bp region from PR2 phage and 500bp region from a different inducible-prophage (PR5), both for genomic DNA (g) and for phages induced and extracted (p) from 39016 WT and ∆PR2 strains. [/fig]

The effect of music on simulated surgical performance: a systematic review Introduction Beneficial effects of music have been described on several cognitive domains, task performance, stress, anxiety and pain. Greater surgical skill is a factor that has been associated with improved patient outcome. The aim of this systematic review is to assess the effect of music on surgical performance. Methods An exhaustive literature search was performed. The following databases were searched: Embase, Medline Ovid, Web of Science, Cochrane CENTAL, PsycINFO Ovid, CINAHL EBSCOhost, ERIC EBSCOhost and Google Scholar. All prospective studies that assessed the effect of a music intervention compared to either another auditory condition or silence on surgical performance were included in a qualitative synthesis. The study was registered in the PROSPERO-database (CRD42018092021). Results The literature search identified 3492 articles of which 9 studies (212 participants) were included. Beneficial effects of music were reported on time to task completion, instrument handling, quality of surgical task performance and general surgical performance. Furthermore, a beneficial effect of music on muscle activation was observed. Conclusion Although beneficial effects of music on surgical performance have been observed, there is insufficient evidence to definitively conclude that music has a beneficial effect on surgical performance in the simulated setting. Future studies should be conducted using greater numbers of participants focusing on a more limited range of tasks, as well as validation in the live operating environment.AbbreviationsEMG Electromyography FLSFundamentals of laparoscopic surgery HRV Heart rate variability OR Operating room or operating theatre PRISMA Preferred reporting items for systematic review and meta analyses TTC Time to task completion * Pim Oomens Music is played during surgery in many operating rooms (ORs) worldwide. A majority of physicians and nurses reported that they listen to music on a regular basis in the OR. Respondents stated that music makes them feel calmer and work more efficient. Rauscher et al. first described beneficial effects of music on spatial task performance. Since then, much has been published on this so-called Mozart effect. A meta-analysis concluded that there is a small but statistically significant beneficial effect of listening to Mozart on task performance. Moreover, this effect can also be observed with other types of music. Beneficial effects of music have been reported on task performance and cognitive abilities in both rodents and humans. Furthermore, anxiolytic and analgesic effects of music during surgery have been observed. Also, stressreductive effects of music in healthcare professionals have been described. Greater surgical skill has been associated with a reduction in postoperative complicationsand high stress levels in the operating theatre can negatively affect surgical performance and team performance. According to a survey, nearly 80% of the responding surgeons experience pain on a regular basis while performing surgery. Since music can improve task performance, reduce stress and has analgesic effects, it could potentially benefit surgical performance and therefore patient outcome. The primary objective of this systematic review is to assess the effect of music on surgical performance. Secondary outcomes are the effect of music on vital parameters, stress and electromyography (EMG). # Methods The study protocol was registered in the PROSPERO database (CRD42018092021). All aspects of the PRISMAstatement were followed. Neither IRB approval nor written informed was necessary to obtain, as this paper is a systematic review. ## Search strategy The databases Embase, Medline Ovid, Web of Science, Cochrane CENTAL, PsycINFO Ovid, CINAHL EBSCOhost, ERIC EBSCOhost and Google Scholar were searched on 1 March 2018 with keywords like "surgery" "surgical skill" "music" and "auditory stimulation". The syntax construction and database search were executed in collaboration with a biomedical information specialist using the exhaustive search method. The full search and syntax is presented in Appendix. Two independent reviewers (PO and VF) identified eligible studies. First, all identified articles were screened by title and abstract. Subsequently, the full text articles were screened to assess if eligibility criteria were matched. Only full text peer-reviewed published articles in the English language were included. Inclusion criteria for this systematic review were prospective studies that assessed the effect of music compared to another auditory condition or to silence on surgical performance. Secondary outcomes were the effect of music on heart rate, blood pressure, stress response and electromyography (EMG). Studies were excluded if multiple concomitant interventions were used. Discrepancies were resolved through mutual discussion or by referring to a senior author (JJ). ## Data collection and quality assessment Data collection was performed independently by two researchers (PO and VF) using customised forms. If data were available in plots or images, data were estimated using the online available data extraction software WebPlotDigitizer (version 4.1). If necessary, authors were contacted to obtain additional data. Risk of bias was assessed using the Cochrane Collaboration's tool for assessing risk of bias . Disagreements between reviewers were resolved through mutual discussion or by referring to a senior author (JJ). # Data analysis The overall group path length and time to task completion (TTC) means were calculated if individual data were presented. Standard error was converted to standard deviation as described in the Cochrane handbook . If a study contained several music interventions, the means and standard deviations of the different music groups were pooled to an approximated mean and standard deviation of the entire group. If several tasks were used to assess surgical performance, approximated means and standard deviations were pooled for the outcomes of time to task completion and path length. If absolute means were presented, mean differences and percentages of mean differences were computed. Only the percentage of improvement was extracted in studies where the task that was used in the intervention group was different from the task in the control group, as parameters such as time to task completion and path length inherently differ between the different tasks. # Results The PRISMA flow diagram of the search strategy is presented in. Initial database searching resulted in 3492 articles (2129 after removal of duplicates). Nine articles (212 participants) were included in this review. An overview of the study characteristics is presented in. All studies assessed surgical performance in a simulated setting. In eight studies, the music intervention was applied during the assessment of the surgical performance. One study applied the music intervention prior to performing the simulation tasks. Motion analysis software was used to assess surgical performance in six studies. Classical music was used as a music intervention in six studies, while preferred music of the participant was used in two studies. All studies used silence or 'no music' as a control intervention. Additional auditory intervention groups consisted of dichotic music, defined as two different types of music applied through each ear, and OR noise. ## Bias assessment Risks of bias of the included studies are presented in Figs. 2, 3. Several studies lacked information to adequately assess all quality domains. Participants could inherently not be blinded due to the nature of the intervention; therefore, risk of performance bias was high in all studies. Detection bias was low in all studies since either motion analysis software or predefined criteria by blinded observers were used to assess surgical performance. The condition (i.e. surgical task performance) was considered to be suitable for a crossover study if subjects were allowed to practice the task first, or if subjects were experienced with the type of task that was performed, or if a learning effect was assessed and was absent. Carryover effect was assessed as low risk of bias in one study as the time between periods was at least 24 h with a median time of 15.5 days. All other crossover studies did not specify the washout period and carryover effect was therefore assessed as unclear risk of bias in these studies. Other bias has been assessed in one study as high risk of bias, since the study design was changed during the experiment. In this study, two music interventions were compared in a crossover study. An additional cohort with no music was added after analysis of the two music interventions. ## Effects of music on time to task completion Eight studies assessed the effect of music on time to task completion . Three studies evaluated whether the relative improvement in time to task completion was higher, when participants repeated a task and were exposed to either music, no music, silence or another auditory intervention. In the study by Wiseman et al., each participant completed a series of three tasks. The music cohort was exposed to classical music or progressive metal music during the second and third task, while the control cohort was not exposed to music. The percentage of improvement was not significantly different between the music groups and control group. In two studies by Conrad et al. with a similar setup, classical music was played throughout the entire experiment during both the first and second task. One of the two studies showed a statistically significant higher percentage of improvement when participants listened to music compared to the improvement measured during silence. The other study did not report a level of significance as the study consisted of only eight participants. Four studies evaluated the mean time to task completion with and without music. Two studies reported a statistically beneficial effects of both preferred music, hiphop and Jamaican music, on time to task completion. One study did not present exact values, but reported no significant difference between groups. ## Effects of music on instrument handling Instrument handling, defined as path length (i.e. the total distance travelled by the instrument tip) or as the percentage of time that the instrument was out of a predefined boundary, was assessed in six studies . Two studies with a similar setup assessed whether improvement of path length was higher when participants repeated a task and were exposed to either classical music, no music, another auditory intervention or silence. One study found that improvement of path length upon repetition was statistically significantly increased during exposure to classical music in comparison to any other control condition. The other study did not report a level of statistical significance, as only eight participants were included. Four studies evaluated the mean path length or percentage out of bound with and without exposure to music. A statistically significant beneficial effect of Jamaican music ]. One study did not present exact values, but reported no significant difference between groups. ## Effects of music on surgical task performance quality The quality of the performed surgical task was assessed in two studies by blinded observers using predefined criteria. Wound, repair graded on a 1-5 scale by blinded plastic surgeons, was performed with significantly better quality when participants listened to their preferred music genre. There was no statistically significant effect of classical music on the quality of a laparoscopically tied knot. ## Effects of music on general surgical task performance Four studies assessed the effect of music on a general score rating surgical task performance. Two studies used the total score, generated by the simulator's built-in software. One study used a validated global rating scale developed by Reznick et al. ]. Shakir et al. used a validated general motion analysis score based on the parameters time to task completion, tremor, extreme movements and overall movement pattern. This general motion analysis score was significantly improved during exposure to preferred music. Significant beneficial effect of classical music on the total score was also observed in simulated intra-ocular surgery. Two studies did not find a statistically significant effect of classical music, activating or deactivating music on the total score. ## Effect of music on vital parameters and muscle activation One study assessed the effect of music on heart rate and heart rate variability (HRV) during surgical performance. Listening to activating music during surgical performance led to an increased heart rate compared to deactivating music and 'no music'. There were no significant differences in HRV. One study assessed the effects of music on muscle activation in the dominant hand using electromyography (EMG) as an indication of muscle fatigue. Mean electromyography activation of the extensor digitorum muscle was significantly reduced when participants listened to any type of researcher-selected music (i.e. classical, hiphop, Jamaican or jazz), while median electromyography frequency did not differ statistically significantly between groups. Music did not have a statistically significant effect on mean electromyography activation of the flexor carpi radialis, but did decrease median electromyography frequency. # Discussion This systematic review provides an overview of the effect of music on surgical performance. Five out of nine studies reported beneficial effects of music on different surgical performance domains. Beneficial effects of music were observed on TTC, instrument handling, task performance qualityand general surgical task performance. Moreover, one study also observed an attenuating effect of music on muscle activation, which can be correlated to muscle fatigue. All included studies assessed the effect of music on surgical skill in a simulated setting. Surgical skill acquired in a simulated setting translates to and correlates with surgical performance in a clinical setting. Greater surgical skill is associated with a lower mortality and complication rate in surgical patients, including surgical site infections, pulmonary complications, readmissions and reoperations. Several studies reported a beneficial effect of music on time to task completion. Prolonged operation duration has been associated with a higher postoperative complication rate and increases medical costs. Therefore, the use of music during surgical procedures could potentially improve patient outcome and reduce costs, as one minute of OR-time is estimated to cost $36-37. Implementing music interventions in training modules might also benefit residents. Simulation based training is an essential part of surgical education, as the American Board of Surgery Graduating requires graduating residents to successfully pass the FLS program (Fundamentals of Laparoscopic Surgery). The type of music that is most beneficial is unclear, but we believe it to be unlikely that a surgeon would listen to music that they dislike. Perhaps the beneficial effect of music on surgical performance is more profound if participants can choose music of their preference. This would coincide with earlier observations where the beneficial effect of music on the surgeon's physiological response was larger under selfselected music compared to researcher-selected music. Out of the nine included studies in this review, two used preferred music of the participants. Both these studies observed statistically significant beneficial effects of music on time to task completion, task performance, quality of repair and on general surgical task performance. Siu et al. used several researcher-selected music genres. Significant beneficial effects of hiphop were observed on time to task completion, hiphop was in the top two favourite genres of 70% of the participants. In another study, a tendency towards improved surgical performance was observed in participants that rated the music as pleasant, compared to unpleasant or to silence. There are several limitations of this review. One limitation is the low number of included studies and participants. While time to task completion was assessed as the primary outcome measure by most studies, it was not reported in a consistent manner. Some studies reported within-subject improvement, while others reported absolute means of the groups. Moreover, the studies contained different simulated tasks. Therefore, no meta-analysis could be performed, and no absolute values (i.e. time reduction in minutes) could be calculated. Other endpoints were reported less frequently. This limits the strength of conclusions that could be drawn. None of the included studies were performed in a live operating environment. There is contradicting evidence with regard to the use of music in the operating theatre. Music has been reported to reduce stress and increase working efficiency in OR-staff. Music has also been reported to impair surgeon's auditory processing and team communication. The majority of anaesthetists generally like music in the operating theatre, but also consider it to be distracting if anaesthesiological problems were to occur. However, in a simulated setting, no adverse effects of music were observed on anaesthetist's psychomotor performance. Many factors can potentially affect surgical performance in a live operating environment, including leadership skills, communication level and cooperation. How music affects all these factors and thus surgical performance in a live operating environment is unclear. Nonetheless, several studies have reported a correlation between improved surgical performance in a simulated setting and performance in the live operating environment. # Conclusion There is no sufficient evidence to definitively determine whether music has a beneficial effect on surgical performance in the simulated setting. However, the results suggest that preferred music of the participant does improve surgical performance in a simulated setting. Future studies should be conducted using greater numbers of participants, participant preferred music, and focusing on a more limited range of tasks. Furthermore the effects of music on surgical team performance and patient outcome should be assessed, in order to answer the question whether music improves surgical performance in the live operating environment. ## Search strategy embase.com (music/de OR 'auditory stimulation'/de OR 'noise'/de OR (music OR musical OR musicotherap* OR (rhythm NEAR/3 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) NEAR/3 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise):ab,ti) AND ('surgical skill'/exp OR 'suture'/de OR 'wound closure'/de OR 'suture technique'/de OR 'surgeon'/exp OR (('motor system'/de OR 'psychomotor performance'/de OR 'motor performance'/de OR 'motor function test'/de OR 'task performance'/de OR 'eye hand coordination'/de OR 'motor activity'/de OR 'motor coordination'/de) AND (surgery/exp OR 'operating room'/exp)) OR (((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) NEAR/3 (surgic* OR surger* OR operating-room* OR operating-theat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR peroperat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci):ab,ti) NOT ([animals]/lim NOT [humans]/lim) AND [english]/lim. ## Medline ovid (music/OR Acoustic Stimulation/OR noise/OR (music OR musical OR musicotherap* OR (rhythm ADJ3 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) ADJ3 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise).ab,ti.) AND (sutures/OR Suture Techniques/OR Wound Closure Techniques/OR exp surgeons/ OR (("Task Performance and Analysis"/OR Psychomotor Performance/OR motor activity/) AND (exp Surgical Procedures, Operative/OR Operating Rooms/)) OR (((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) ADJ3 (surgic* OR surger* OR operating-room* OR operating-theat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR per-operat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci).ab,ti.) NOT (exp animals/ NOT humans/) AND english.la. ## Psycinfo ovid (music/OR Auditory Stimulation/OR noise effects/OR (music OR musical OR musicotherap* OR (rhythm ADJ3 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) ADJ3 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise).ab,ti.) AND (exp surgeons/OR (((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) ADJ3 (surgic* OR surger* OR operating-room* OR operating-theat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR per-operat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci). ab,ti.) NOT (exp animals/NOT humans/) AND english.la. ## Cinahl ebscohost (MH music OR MH Acoustic Stimulation OR MH noise OR TI (music OR musical OR musicotherap* OR (rhythm N2 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) N2 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise) OR AB (music OR musical OR musicotherap* OR (rhythm N2 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) N2 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise)) AND (MH sutures OR MH Suture Techniques OR MH surgeons + OR ((MH "Task Performance and Analysis" OR MH Psychomotor Performance + OR MH motor activity) AND (MH Operating Rooms OR MH Surgery, Operative +)) OR TI (((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) N2 (surgic* OR surger* OR operating-room* OR operating-theat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR per-operat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci) OR AB (((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) N2 (surgic* OR surger* OR operating-room* OR operating-theat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR per-operat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci)) NOT (MH animals + NOT humans +) AND LA (english). ## Eric ebscohost (MH music OR TI (music OR musical OR musicotherap* OR (rhythm N2 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) N2 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise) OR AB (music OR musical OR musicotherap* OR (rhythm N2 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) N2 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise)) AND (TI (((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) N2 (surgic* OR surger* OR operating-room* OR operating-theat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR peroperat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci) OR AB (((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) N2 (surgic* OR surger* OR operating-room* OR operating-theat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR peroperat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci)) NOT (MH animals + NOT humans +) AND LA (english). ## Cochrane central ((music OR musical OR musicotherap* OR (rhythm NEAR/3 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) NEAR/3 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise):ab,ti) AND ((((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) NEAR/3 (surgic* OR surger* OR operating-room* OR operatingtheat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR per-operat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci):ab,ti). ## Web of science TS = (((music OR musical OR musicotherap* OR (rhythm NEAR/2 (perception* OR accompan*)) OR melod* OR ((auditor* OR acoustic*) NEAR/2 (distract* OR condition* OR stress* OR relax* OR stimulat*)) OR noise)) AND ((((motor* OR psychomotor* OR performan* OR abilit* OR function* OR skill* OR train* OR entrain* OR education* OR learn* OR simulat* OR improv* OR sequence* OR process* OR interaction* OR coordinat* OR task*) NEAR/2 (surgic* OR surger* OR operating-room* OR operatingtheat* OR laparoscop* OR perioperat* OR peroperat* OR peri-operat* OR per-operat*)) OR surgeon* OR stitch* OR sutur* OR laparoscop* OR davinci OR da-vinci))) AND LA = (english). ## Google scholar music|musical||"auditory|acoustic distraction|stress|relaxati on"|noise surgeon|"surgical skills|tasks".

Association of education level with diabetes prevalence in Latin American cities and its modification by city social environment Background Diabetes prevalence continues to increase in urban areas of low-income and middle-income countries (LMIC). Evidence from high-income countries suggests an inverse association between educational attainment and diabetes, but research in LMIC is limited. We investigated educational differences in diabetes prevalence across 232 Latin American (LA) cities, and the extent to which these inequities vary across countries/ cities and are modified by city socioeconomic factors. Methods Using harmonised health survey and census data for 110 498 city dwellers from eight LA countries, we estimated the association between education and diabetes. We considered effect modification by city Social Environment Index (SEI) as a proxy for city-level development using multilevel models, considering heterogeneity by sex and country. Results In women, there was an inverse dose-response relationship between education and diabetes (OR: 0.80 per level increase in education, 95% CI 0.75 to 0.85), consistent across countries and not modified by SEI. In men, Argentina, Brazil, Colombia, Chile and Mexico showed an inverse association (pooled OR: 0.92; 95% CI 0.86 to 0.99). Peru, Panama and El Salvador showed a positive relationship (pooled OR 1.24; 95% CI 1.04 to 1.49). For men, these associations were further modified by city-SEI: in countries with an inverse association, it became stronger as city-SEI increased. In countries where the association was positive, it became weaker as city-SEI increased. Conclusion Social inequities in diabetes inequalities increase as cities develop. To achieve non-communicable disease-related sustainable development goals in LMIC, there is an urgent need to develop policies aimed at reducing these educational inequities. # Introduction According to the WHO, diabetes prevalence has doubled over the past 30 years reaching 8.4% in 2014, with 75% of the cases occurring in lowincome and middle-income countries (LMIC).Although diabetes risk factors such as body mass index appear to be increasing in both rural and urban areas, 2 in many LMIC urban living may be especially conducive to diabetes through living and working environments; promoting sedentary work, processed foods consumption, and automobile transportation, all of which may impact risk factors for diabetes. [bib_ref] The obesity transition: stages of the global epidemic, Jaacks [/bib_ref] [bib_ref] Diabetes in South and Central America: an update, Aschner [/bib_ref] [bib_ref] Understanding the rise of cardiometabolic diseases in low-and middle-income countries, Miranda [/bib_ref] In 2012, there were 62 million people with diabetes in Latin America and the Caribbean (LAC), this number is predicted to increase to 109 million by 2040. [bib_ref] IDF diabetes atlas: global estimates for the prevalence of diabetes for 2015..., Ogurtsova [/bib_ref] The LAC region is highly urbanised, with a substantial proportion of persons with diabetes residing in cities.Evidence from highincome countries (HIC) suggests that diabetes is strongly linked to socioeconomic disadvantage 8 but studies of the social patterning of diabetes in large and growing cities of LMIC remain scant. [bib_ref] Socioeconomic status and non-communicable disease behavioural risk factors in low-income and lower-middle-income..., Allen [/bib_ref] Some evidence suggests that social gradients in diabetes are larger in more urbanised LMIC or in more urbanised regions within LMIC. [bib_ref] Association between urbanisation and type 2 diabetes: an ecological study, Gassasse [/bib_ref] [bib_ref] Socioeconomic gradients in chronic disease risk factors in middle-income countries: evidence of..., Fleischer [/bib_ref] [bib_ref] Educational inequalities in obesity among Mexican women: time-trends from 1988 to 2012, Ferrer [/bib_ref] Several factors may contribute to diabetes inequities in urban areas. Urban living (and changes in income and work associated with it) has been linked to more processed foods consumption, and higher prevalence of drinking, smoking and sedentary lifestyles. This increase initially occurs among the wealthiest populations but later transitions into a disproportionate increase in disadvantaged populations. [bib_ref] Dissonant health transition in the states of Mexico, 1990-2013: a systematic analysis..., Gómez-Dantés [/bib_ref] [bib_ref] The nutrition transition in Mexico 1988-2016: the role of wealth in the..., Pérez-Ferrer [/bib_ref] [bib_ref] Independent effects of income and education on the risk of obesity in..., Monteiro [/bib_ref] [bib_ref] Ultra-processed foods and the nutrition transition: global, regional and national trends, food..., Baker [/bib_ref] Despite the importance of diabetes in urban areas and the documented presence of stronger diabetes inequities in more urbanised areas, [bib_ref] Socioeconomic status and non-communicable disease behavioural risk factors in low-income and lower-middle-income..., Allen [/bib_ref] [bib_ref] Association between urbanisation and type 2 diabetes: an ecological study, Gassasse [/bib_ref] [bib_ref] Socioeconomic gradients in chronic disease risk factors in middle-income countries: evidence of..., Fleischer [/bib_ref] no prior research has examined how specific features of urban areas affect the social patterning of diabetes in cities. Similarly, to what has been observed for countries, cities that have higher incomes, better infrastructure (including housing), and higher proportion of people with higher education levels may also paradoxically exhibit greater educational inequalities in chronic disease risk, because beneficial resources and environments may be unequally distributed. Understanding and documenting educational inequities in diabetes in LMIC and its variations across different urban environments is necessary to develop effective interventions to reduce the prevalence of diabetes across rapidly growing cities of LMIC. Using a harmonised dataset of individual and city-level data for 232 cities in eight Latin American countries, we investigated educational differences in diabetes prevalence by gender, whether these inequities vary across countries and cities, and the extent to which they are modified by city social environment. # Methods Data are from the Salud Urbana en America Latina Project project, which has compiled and harmonised health, social and built-environment data from 371 large cities (population ≥100 000 in 2010) from 11 countries. [bib_ref] Building a data platform for Cross-Country urban health studies: the SALURBAL study, Quistberg [/bib_ref] These analyses are based on a 232 cities subset from 8 countries with available survey data on diabetes: Argentina, Brazil, Chile, Colombia, El Salvador, Mexico, Peru and Panama. Surveys were designed for chronic disease surveillance using random probabilistic household sampling. Sample sizes for countries ranged from 1333 to 30 425 participants. Survey details are provided in online supplemental table 1. The main outcome was self-reported diabetes in adults 25 years or older. Survey participants were classified as having diabetes if they reported being given a diabetes diagnosis by a healthcare provider. Female participants who reported a diagnosis of gestational diabetes were not considered to have diabetes, except in the case of Argentina and Panama because information on pregnancy status during the time of diagnosis was unavailable. The two key exposures investigated were city Social Environment Index (SEI) and individual-level education, a proxy of individual-level SES. The SEI combines four variables representing various aspects of the city social environment: water access (% households with access to piped water); sanitation (% households with access to a municipal sewage network); overcrowding (% households with >3 people per room); and primary education completion (% population ≥25 years with at least completed primary education). This measure was created by summing the standardised Z-scores of the four variables (overcrowding was reverse coded). The SEI is used as a global metric of city social environment since it captures various aspects of city-living conditions. It has been shown to be related to differences in life expectancy across cities in the sample. [bib_ref] Inequalities in life expectancy in six large Latin American cities from the..., Bilal [/bib_ref] For descriptive analyses, the entire sample was divided into SEI tertiles (low, medium, high). Individual-level education was obtained from survey reports. Data were harmonised using the Integrated Public Use Microdata Series 21 harmonisation definitions and categorised into the following categories: (1) less than primary: individuals with no education or less than completed primary education; (2) primary: individuals who completed primary education, but with incomplete secondary education; (3) secondary: individuals with complete secondary education, complete non-university postsecondary education (eg, technical school), or with incomplete university education; (4) university or higher: individuals who completed a university degree or with complete/incomplete graduate studies. Because descriptive analyses suggested a linear pattern in the association between education categories and diabetes, education was examined as a continuous variable ranging from 0 to 3 reflecting increasing education categories. Age (range 25-97 years) and sex were included as covariates. # Statistical analysis Descriptive statistics for all predictors are presented by city-SEI tertiles and diabetes status. We estimated age-adjusted proportions by education categories and gender separately for each country using Poisson regression models. We inspected patterns (ie, whether they were approximately graded or exhibited thresholds) as well as major differences across countries in the nature of the association (eg, inverse, none, positive). We estimated the association of city-SEI and individuallevel education with diabetes using two-level mixed logistic regression models stratified by sex with individuals nested within cities, adjusting for country fixed effects. We included a random intercept for each city and a random slope for education to allow education gradients to differ between cities. Model 1 included the two key predictors city-SEI and individual-level education, as well as age. Model 2 included country fixed effects. In model 3, we included interactions between education and country fixed effects to determine if there were differences in education gradients by country. If the interaction was statistically significant, we further examined heterogeneity by grouping countries into two groups based on educational patterns observed in descriptive analyses (group 1, displaying an inverse crude association: Argentina, Brazil, Chile, Colombia, Mexico; group 2, displaying a positive crude association: Peru, Panama, El Salvador) and including an appropriate interaction term. To test our hypothesis that city-SEI modified educational differences, we fitted model 4 which added an interaction term between city-SEI and individual-level education (both as continuous variables). Using this model, we estimated the effect of a one-level increase in education for people living in cities at the 10th, 50th and at the 90th percentiles of city-SEI. We plotted the predicted probabilities resulting from the model according to education level by city-SEI. All analyses were stratified by sex considering previous evidence suggesting that the effects of social environment and education on non-communicable diseases (NCDs) vary by sex. [bib_ref] Sex differences in the relationship between socioeconomic status and cardiovascular disease: a..., Backholer [/bib_ref] For the models, city-SEI was standardised to a mean of 0 and a SD of 1. We specified a level of significance of 0.05 and all analysis was conducted using SAS 9.4 and R V.3.6.1 software. # Results Our sample included 110 498 survey participants in 232 cities from eight Latin American countries. There was a median of 300 individuals per city (IQR 120-600). [fig_ref] Table 1: City-level and individual-level characteristics by city-level SEI tertile SEI-education interaction and was... [/fig_ref] shows city and individual-level characteristics according to city-SEI. The full sample had a median age of 43 years and 59% were women. The highest percentage of survey participants lived in Brazil and Mexico (27.5% and 21.2%, respectively), while El Salvador and Chile had the lowest (1.2% and 2.2%, respectively). Age and gender did not differ across SEI tertiles. The percentage of people with diabetes was 7%, 9% and 8%, respectively, at the lowest, middle and highest city-SEI tertile. Higher SEI cities tended to have a larger proportion of inhabitants within higher educational categories. Persons with diabetes lived in cities with a slightly higher SEI were older and more concentrated in the lower education categories than those without diabetes (online supplemental table 2). [fig_ref] Figure 1: Age-adjusted percentage of people with diabetes by country and gender according to... [/fig_ref] shows the age-adjusted proportion of respondents with diabetes by education level by sex. In women, there was a clear inverse association between diabetes and education for all countries except Peru; additionally, in Argentina, Brazil and Mexico there was a dose-response pattern. In men, there was an inverse association in Argentina, Brazil and Chile without a consistent dose-response relationship, but no clear pattern was observed in Mexico and Colombia, although a slightly higher prevalence of diabetes was observed in the lower educational categories. In men living in Peru, Panama and El Salvador, there was a positive association with a doseresponse pattern: the higher the education level, the higher the diabetes prevalence. ## Original research Given the patterns observed in figure 1, we tested for multiplicative interactions between education and country. The global test for education-country interaction was statistically significant in men (p=0.005) but not in women (p=0.4). We also tested for heterogeneity across sets of countries with different patterns based on visual inspection of figure 1. In women, we found no evidence that associations differed significantly in Peru (p for interaction=0. [bib_ref] Socioeconomic status is associated with global diabetes prevalence, Xu [/bib_ref] or Chile, Colombia, Panama and El Salvador (p=0.2) compared with Argentina, Brazil and Mexico. Consequently, we estimated associations of education with diabetes pooling across all countries but adjusting for country fixed effects. In men, we found that associations differed significantly in Peru, El Salvador and Panama compared with the other countries (p for interaction=0.002). Hence, we provide two estimates of education associations: one for Colombia, Mexico, Argentina, Brazil and Chile, and another for Peru, El Salvador and Panama. [fig_ref] Table 2: ORs of diabetes associated with individual-level education and city-SEI by sex [/fig_ref] shows the ORs of diabetes associated with individuallevel education and city-SEI. In women, higher individual-level education was strongly and inversely associated with lower odds of diabetes, after adjusting for country fixed effects (OR 0.80; 95% CI 0.75 to 0.85). No associations were observed for city-SEI. The random effects for the intercept and education slope remained significant even after adjusting for country, providing evidence of residual variation in diabetes and the effect of education across cities. In men, after adjusting for country fixed effects higher individual-level education was associated with lower odds of diabetes in Argentina, Brazil, Chile, Colombia and Mexico (0R 0.92; 95% CI 0.86 to 0.98). For Peru, Panama and El Salvador, the association was reversed, the odds of diabetes increased by 24% per one-unit higher education level (OR 1.24; 95% CI 1.04 to 1.49), after adjusting for city-SEI, age and country fixed effects. City-SEI was not associated with diabetes. The random intercept remained significant suggesting of residual variability in diabetes between cities. The variance of the random slope for education was no longer statistically significant when we introduced the interaction between education and country. [fig_ref] Table 3: ORs [/fig_ref] shows associations of individual-level education with diabetes for cities with different levels of SEI. In women, there was no evidence of SEI-education interaction (p=0.834) and the odds of diabetes were reduced by 20% for each one-unit increase in education level across the different SEI levels. The random slope for education remained statistically significant suggesting residual variation in the effect of education across cities. In men, there was a statistically significant interaction (p=0.019) between education and city-SEI. We present the OR of diabetes associated with education separately for the two sets of countries given qualitative differences in the directions of the associations between education and diabetes. Men in Argentina, Brazil, Chile, Colombia and Mexico had an inverse association [fig_ref] Figure 2: Predicted probabilities of diabetes based on the multilevel logistic regression models to... [/fig_ref] depicts these patterns graphically for predicted probabilities derived from the model with interactions. # Discussion We used survey data for 232 large cities in eight Latin American countries to characterise educational differences in diabetes prevalence and examine the extent to which these inequities varied across countries and cities. We also examined whether city Age adjusted percentage of women with diabetes by education level Bold values have a p value<0.05. *All models were adjusted for individual age. Education is modelled as a continuous variable, a one-unit increase reflects one higher level of education, for example, less than primary to primary complete. City Social Environment Index (SEI) was standardised to a mean of 0 and an SD of 1. The OR is estimated for a 1 SD (0.58703) difference in SEI. †Model 2 is adjusted for country fixed effects. ‡Model 3 includes an interaction between education and different country groups only for men (p for interaction = 0.002). Combinations of the main effect of education and the interaction coefficient were used to derive estimates for different countries. ## Less than primary University or more ## Less than primar University or more y ## Original research social environment modified the relationship between education and diabetes. In women, we found a clear educational gradient; women with higher education levels had a lower proportion of diabetes, with a dose-response pattern for all countries (except possibly Peru). Furthermore, this association was not modified by city-SEI. In men, the association of education with diabetes varied across countries; in some countries, there was an inverse association while in other countries there was no association or the opposite association (higher proportion of diabetes in men with higher education). Moreover, this association was modified by city-SEI such that an inverse association emerged (or a positive association weakened) as the city-SEI improved. Evidence from HIC consistently shows a higher prevalence of diabetes in people with lower education levels compared with higher education and this association is consistently stronger in women. [bib_ref] Sex differences in the relationship between socioeconomic status and cardiovascular disease: a..., Backholer [/bib_ref] [bib_ref] Socioeconomic inequalities in diabetes mellitus across Europe at the beginning of the..., Espelt [/bib_ref] Limited data from LMIC suggest heterogeneity in social gradients. We found that among Latin American women in urban areas, the association of education with diabetes follows the same pattern found in HIC. [bib_ref] Sex differences in the relationship between socioeconomic status and cardiovascular disease: a..., Backholer [/bib_ref] For men, the pattern is not constant across countries. In the case of Peru, El Salvador and Panama, we found a higher prevalence of diabetes among men with higher education. Other research from HIC and some Latin American countries has documented stronger inverse educational or socioeconomic gradients in obesity, diabetes, and NCDs in women but weaker or opposite gradients in men. [bib_ref] Educational inequalities in obesity among Mexican women: time-trends from 1988 to 2012, Ferrer [/bib_ref] Education may be more beneficial to women because they may lack other resources such as earnings, power and authority. [bib_ref] Gender and the health benefits of education, Ross [/bib_ref] Social norms regarding weight, physical activity and diet (all risk factors for diabetes) maybe more strongly patterned by education in women than in men resulting in differential associations of diabetes with education by sex. [bib_ref] Gender-related differences in the association between socioeconomic status and self-reported diabetes, Tang [/bib_ref] In our study, country modified educational gradients in men but not in women. The countries in which we observed that higher education was associated with more diabetes in menincluding Peru and El Salvador-are the countries with the lowest income per capita in our sample.These findings are consistent with prior work showing that country economic development modifies social gradients in chronic disease risk. In the early stages of the nutritional transition, men with higher socioeconomic status tend to be less physically active and consume more processed foods with high content of fat and salt, compared with men in lower socioeconomic status. As countries develop, this pattern reverses and those with higher income and education have healthier diets and behaviours. Why this modification of educational gradients by country was more evident in men than in women is a question that deserves additional research. Our sample includes 232 cities from eight Latin American countries, with large heterogeneity in social environments. Models were adjusted for age, country fixed effects, and education and SEI main effects. *The model for men also includes the interaction between individual education and country. Thus, we expected our results to vary between cities. No effect modification was observed in women. In men, we found that city-SEI modified the association of education with diabetes. In countries with an inverse association, it became significant and was stronger as city-SEI increased. In countries where the association was positive, it became weaker as city-SEI increased. These results mimic the country-level differences but across cities within a country. These findings are consistent with a recent study in India which found that the positive association between diabetes and individual SES in less developed districts became weaker or inverse in districts with higher levels of development. [bib_ref] The interaction between district-level development and individual-level socioeconomic gradients of cardiovascular disease..., Jung [/bib_ref] Our results highlight the importance of considering the different social and economic environments within a region and how the effects of individual-level factors may be modified by environmental conditions. An important limitation of our study is the use of self-report survey data for the ascertainment of diabetes status. This may have led to underestimates in groups with less access to healthcare and may have consequently resulted in underestimates of educational gradients. Gender differences in access to healthcare, such as prenatal care identification of high-risk women, and pattering of healthcare access by SES could also have impacted the gender differences that we observed. The year of the surveys and census measures are not fully aligned across countries, although some stability in the census-based measures of population characteristics can be assumed. [bib_ref] The obesity transition: stages of the global epidemic, Jaacks [/bib_ref] Because our focus was on estimating associations rather that prevalences for specific cities we did not use survey weights; however, variables related to the weights were included as adjustment factors. Our results are cross sectional and cannot be used to draw causal inferences but still provide valuable descriptive information relevant to policy. Major strengths of our study include the large sample of individuals (110 498) and cities (232) representing a significant proportion of the urban population of Latin America, the harmonised dataset, and the availability of city-specific information absent from past work. Our models are adjusted for country fixed effects that would account for any unmeasured country factors such as differences in healthcare and education systems across countries. In summary, we found strong educational gradients in diabetes in women living in Latin American cities, regardless of city socioeconomic development. Findings in men suggest that inverse gradients may emerge as country and city socioeconomic development increase. Our results show that social gradients varied and modified by country and local contexts. Considering the increasing trends of diabetes and the high urbanisation in Latin America, it is paramount to develop policies aimed at reducing social inequities in diabetes in cities. These policies need to be sensitive to the ways in which inequities in diabetes manifest in different social and economic contexts and how they may vary across countries and cities. Targeting socioeconomic and gender inequities in cities will be critical to achieving the sustainable development goals related to reducing NCDs in LMIC. Correction notice This article has been corrected since it first published. The 7th affiliation has been corrected. ## Twitter ariela braverman-bronstein @aribravs Acknowledgements The authors acknowledge the contribution of all SALURBAL project team members. For more information on SALURBAL and to see a full list of investigators, see https:// drexel. edu/ lac/ salurbal/ team/. SALURBAL acknowledges the contributions of many different agencies in generating, processing, facilitating access to data or assisting with other aspects of the project. Visit https:// drexel. edu/ lac/ data-evidence for a complete list of data sources. Competing interests None declared. ## Contributors ## Patient consent for publication not required. Ethics approval The SALURBAL study protocol was approved by the Drexel University IRB (ID#1612005035) and by appropriate site-specific IRBs. Provenance and peer review Not commissioned; externally peer reviewed. Data availability statement Data are available upon reasonable request. The SALURBAL project welcomes queries from anyone interested in learning more about its dataset and potential access to data. To learn more about SALURBAL's dataset, visit https:// drexel. edu/ lac/ or contact the project at salurbal@ drexel. edu. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. ## Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, What is already known on this subject ► Previous evidence from high-income countries reports an inverse association of diabetes with individual-level socioeconomic status; however, studies of the social patterning of diabetes in low-income and middle-income countries (LMIC) are scant. Some evidence suggests that social gradients in diabetes are larger in LMIC that are more urbanised compared with less urbanised ones and that social gradients also vary within regions of LMICs, with larger social gradients observed in more compared with less urbanised areas. However, no studies have examined inequities in a systematic fashion across the growing cities of LMIC or how these gradients may vary across countries or be modified by city characteristics. ## What this study adds ► This is the first study to comprehensively examine social gradients in diabetes across the cities of Latin America. We found that in women there was a clear educational gradient in diabetes: women with higher education levels had a lower proportion of diabetes, with a dose-response pattern for all countries. Furthermore, this association was not modified by city social environment. In men, the association of education with diabetes varied across countries and this association was modified by city social environment as such that an inverse association emerged (or a positive association disappeared) as the city socioeconomic conditions improved. It is paramount to develop policies aimed at reducing social inequities in diabetes which can be expected to increase over time as cities continue to grow and develop unequally. ## Original research and indication of whether changes were made. See: https:// creativecommons. org/ licenses/ by/ 4. 0/. ## Orcid ids Ariela Braverman-Bronstein http:// orcid. org/ 0000-0002-9999-808X Andrés Trotta http:// orcid. org/ 0000-0003-4181-1494 [fig] Figure 1: Age-adjusted percentage of people with diabetes by country and gender according to their individual education level. Age was categorised into three groups: 25-39, 40-64, and 65 and older. These categories were further included in Poisson regression models to estimate diabetes prevalence by education levels. [/fig] [fig] Figure 2: Predicted probabilities of diabetes based on the multilevel logistic regression models to assess the effect modification of education level by city-Social Environment Index (SEI). Education level: 0=less than primary; 1=primary; 2=secondary; 3=university or more. City-SEI values are based on the standardised variable used in the model so the range of values differs from the on presented in table 1. [/fig] [table] Table 1: City-level and individual-level characteristics by city-level SEI tertile SEI-education interaction and was not statistically significant. [/table] [table] Table 2: ORs of diabetes associated with individual-level education and city-SEI by sex [/table] [table] Table 3: ORs (95% CI) of diabetes associated with education for cities with different levels of the Social Environment Index (SEI) and residual education slope variance across cities [/table]

The association of smoking and socioeconomic status on cutaneous melanoma: a population‐based, data‐linkage, case–control study† Background Previous studies have identified an inverse association between melanoma and smoking; however, data from population-based studies are scarce. Objectives To determine the association between smoking and socioeconomic (SES) on the risk of development of melanoma. Furthermore, we sought to determine the implications of smoking and SES on survival. Methods We conducted a population-based case-control study. Cases were identified from the Welsh Cancer Intelligence and Surveillance Unit (WCISU) during 2000-2015 and controls from the general population. Smoking and SES were obtained from data linkage with other national databases. The association of smoking status and SES on the incidence of melanoma were assessed using binary logistic regression. Multivariate survival analysis was performed on a melanoma cohort using a Cox proportional hazard model using survival as the outcome. Results During 2000-2015, 9636 patients developed melanoma. Smoking data were obtained for 7124 (73Á9%) of these patients. There were 26 408 controls identified from the general population. Smoking was inversely associated with melanoma incidence [odds ratio (OR) 0Á70, 95% confidence interval (CI) 0Á65-0Á76]. Smoking was associated with an increased overall mortality [hazard ratio (HR) 1Á30, 95% CI 1Á09-1Á55], but not associated with melanoma-specific mortality. Patients with higher SES had an increased association with melanoma incidence (OR 1Á58, 95% CI 1Á44-1Á73). Higher SES was associated with an increased chance of both overall (HR 0Á67, 95% CI 0Á56-0Á81) and disease-specific survival (HR 0Á69, 95% CI 0Á53-0Á90). Conclusions Our study has demonstrated that smoking appeared to be associated with reduced incidence of melanoma. Although smoking increases overall mortality, no association was observed with melanoma-specific mortality. Further work is required to determine if there is a biological mechanism underlying this relationship or an alternative explanation, such as survival bias. ## Summary Background Previous studies have identified an inverse association between melanoma and smoking; however, data from population-based studies are scarce. Objectives To determine the association between smoking and socioeconomic (SES) on the risk of development of melanoma. Furthermore, we sought to determine the implications of smoking and SES on survival. Methods We conducted a population-based case-control study. Cases were identified from the Welsh Cancer Intelligence and Surveillance Unit (WCISU) during 2000-2015 and controls from the general population. Smoking and SES were obtained from data linkage with other national databases. The association of smoking status and SES on the incidence of melanoma were assessed using binary logistic regression. Multivariate survival analysis was performed on a melanoma cohort using a Cox proportional hazard model using survival as the outcome. Results During 2000-2015, 9636 patients developed melanoma. Smoking data were obtained for 7124 (73Á9%) of these patients. There were 26 408 controls identified from the general population. Smoking was inversely associated with melanoma incidence [odds ratio (OR) 0Á70, 95% confidence interval (CI) 0Á65-0Á76]. Smoking was associated with an increased overall mortality [hazard ratio (HR) 1Á30, 95% CI 1Á09-1Á55], but not associated with melanoma-specific mortality. Patients with higher SES had an increased association with melanoma incidence (OR 1Á58, 95% CI 1Á44-1Á73). Higher SES was associated with an increased chance of both overall (HR 0Á67, 95% CI 0Á56-0Á81) and disease-specific survival (HR 0Á69, 95% CI 0Á53-0Á90). Conclusions Our study has demonstrated that smoking appeared to be associated with reduced incidence of melanoma. Although smoking increases overall mortality, no association was observed with melanoma-specific mortality. Further work is required to determine if there is a biological mechanism underlying this relationship or an alternative explanation, such as survival bias. What does this study add? - Our large study identified an inverse association between smoking status and melanoma incidence. - Although smoking status was negatively associated with overall disease survival, no significant association was noted in melanoma-specific survival. - Socioeconomic status remains closely associated with melanoma. Although higher socioeconomic populations are more likely to develop the disease, patients with lower socioeconomic status continue to have a worse prognosis. Although there is a wealth of knowledge on the association of melanoma with risk factors such as ultraviolet light exposure, skin type and genetics, 1 the relationship between tobacco smoke and melanoma is less clear. Tobacco smoke is a type 1 carcinogen, associated with 18 types of cancer.Song et al. [bib_ref] Smoking and risk of skin cancer: a prospective analysis and a meta-analysis, Song [/bib_ref] reported a moderate inverse association between melanoma and smoking in a meta-analysis of two cohort studies. This association was observed in both ex-smokers and current smokers in men, but not women. A larger meta-analysis, including 23 studies, reported a similar inverse association. [bib_ref] Smoking is inversely related to cutaneous malignant melanoma: results of a meta-analysis, Li [/bib_ref] Both papers reported significant limitations, notably publication bias because of selective reporting in the published studies. Furthermore, confounding variables were not included in the analysis. A recent, prospective cohort study has further explored the association. After adjusting for potential confounding factors, no association was observed between current smoking and melanoma [odds ratio (OR) 1Á01, 95% confidence interval (CI) 0Á64-1Á61]. [bib_ref] Smoking and cutaneous melanoma: findings from the QSkin Sun and Health Cohort..., Dusingize [/bib_ref] Although the study addressed the aforementioned limitations by adjusting for confounding factors, the study was significantly underpowered; only a small proportion of the cohort developed melanoma and the average follow-up duration was short (3Á5 years). The relationship between socioeconomic status and melanoma, on the other hand, is well established in the literature, with research dating back to the 1980s. 6,7 Those in higher income or higher educational groups are at an increased risk of developing melanoma, attributed to greater exposure to lifestyle factors, such as sun holidays and tanning bed use. [bib_ref] Socioeconomic status and cutaneous malignant melanoma in Northern Europe, Idorn [/bib_ref] However, once diagnosed, those with a lower socioeconomic status have a worse prognosis, a finding seen across multiple jurisdictions with different healthcare systems. [bib_ref] Socioeconomic status and cutaneous malignant melanoma in Northern Europe, Idorn [/bib_ref] Understanding and addressing this worsened prognosis is therefore a clear public health priority. [bib_ref] Slip, slop, slap, seek, slide -is the message really getting across?, Buchanan [/bib_ref] [bib_ref] Population and agegroup trends in weekend sun protection and sunburn over two..., Makin [/bib_ref] In this paper we describe the largest study investigating the association of smoking and melanoma published to date. We have used the power of routinely collected data to overcome limitations of previous studies and investigate the prognostic implications of smoking in this patient cohort. Furthermore, we sought to investigate the association of socioeconomic status on the incidence and survival of melanoma. # Patients and methods The described study has been reported in accordance with the Reporting of studies Conducted using Observational Routinely collected health Data (RECORD) statement (see [fig_ref] Table 1: List of databases used and their description Database Description Annual District Death... [/fig_ref] in the Supporting Information). [bib_ref] The REporting of studies Conducted using Observational Routinely-collected health Data (RECORD) Statement, Benchimol [/bib_ref] The study was conducted in two stages. In stage one, a case-control study was performed to assess the relationship between smoking and the development of melanoma. In stage two, a cohort study was conducted to determine the association between smoking and survival within the melanoma cohort [fig_ref] Fig 1: Study design [/fig_ref]. # Overview of methods Analyses of primary and secondary care National Health Service data and national administrative data for 2000-2015 in Wales, U.K. (population 3Á1 million) were performed. In instances where relevant data were unavailable from a single source, multiple datasets were linked. Data were retrieved from six national databases [fig_ref] Table 1: List of databases used and their description Database Description Annual District Death... [/fig_ref]. In Wales, populationlevel de-identified person-based health and socioeconomic administrative datasets are collated and linked within the Secure Anonymised Information Linkage (SAIL) databank. [bib_ref] The SAIL Databank: building a national architecture for e-health research and evaluation, Ford [/bib_ref] [bib_ref] A case study of the Secure Anonymous Information Linkage (SAIL) Gateway: a..., Jones [/bib_ref] [bib_ref] The SAIL databank: linking multiple health and social care datasets, Lyons [/bib_ref] Robust policies, structures and controls are in place to protect privacy through a reliable matching and anonymization process, achieved in conjunction with the NHS Wales Informatics Service using a split-file multiple-encryption approach described in detail in previous published work. [bib_ref] A case study of the Secure Anonymous Information Linkage (SAIL) Gateway: a..., Jones [/bib_ref] ## Cases In Wales, all patients with a diagnosis of melanoma are recorded in the Welsh Cancer Intelligence and Surveillance Unit (WCISU) register. Cases were identified from WCISU using International Classification of Disease 10 (ICD-10) codes C43Á0-C43Á9 and morphology codes according to the International Classification of Diseases for Oncology (ICDO-3) 8720-8790.Patients with melanoma in situ were not included in the study as either cases or controls. Demographic information was assessed at the date of diagnosis. Melanoma-specific variables (tumour location, stage and morphology) were assessed at the date of diagnosis. ## Controls Four sets of general population controls were randomly selected from the Welsh Demographic Service Dataset. Controls were not matched to cases. Both cases and controls needed to be alive and resident in Wales on the date of melanoma diagnosis. To increase the power of the study we aimed to have four controls for every case. [bib_ref] Compared to what? Finding controls for casecontrol studies, Grimes [/bib_ref] ## Smoking status Self-reported smoking status, for cases and controls were obtained from the Welsh Longitudinal General Practice (WLGP) data, as recorded during patients' consultations with their general practitioner in primary care, using Read codes that have been previously validated [bib_ref] Development of an algorithm for determining smoking status and behaviour over the..., Atkinson [/bib_ref] (for lifelong nonsmokers), ex-smoker (for those that had previously smoked) or current smokers. The smoking assessment window extended from the melanoma diagnosis date to 6 months prior. Where serial assessments were available, the smoking record most recent to the diagnosis was selected. Where 'nonsmoker' was recorded, the WLGP dataset was explored to establish whether the individual had previously been classified as a smoker. In such circumstances, the individual was classed as an ex-smoker. ## Socioeconomic status Socioeconomic status was measured using the Welsh Index of Multiple Deprivation (WIMD) version 2001, a measure based on the Index of Multiple Deprivation and used as the official measure of socioeconomic status for the Welsh Government.Individual scores are based upon a person's postal address. Wales is divided into 1896 lower-layer super-output areas (LSOAs) following the 2001 Census, each consisting of approximately 1600 people. The WIMD scores for each LSOA are calculated from weighted scores from eight domains of socioeconomic status (income, employment, health, education, access to services, community safety, physical environment and housing socioeconomic status). Each LSOA in Wales has been ranked according to its WIMD score and grouped into quintiles, with quintile five being the highest socioeconomic status and one being the lowest. ## Mortality data Data relating to mortality, including cause of death for the melanoma cohort were obtained from the Annual District Death Extract dataset, which contains the diagnostic codes listed on patient's death certificates, held within the SAIL Databank. ## Charlson comorbidity index The Charlson Comorbidity Index is a widely used measure of comorbidity. An overall score is calculated from a list of conditions, each of which has been allocated a weight of between one and six based upon its adjusted relative risk of 1-year mortality. 20 # Ethical approval Study approval was granted by the SAIL Databank independent Information Governance Review Panel (project 0593). Data held within the SAIL Databank are made available to researchers in an anonymized format and are therefore not subject to data protection legislation. SAIL follows all relevant legislative and regulatory frameworks in using population data for research. # Statistical analysis ## Case-control (stage one) Descriptive statistics were used to characterize the melanoma cases and controls by smoking status and stage at diagnosis (cases only). An unconditional binary logistic regression model was used to calculate ORs with 95% CIs for the association with melanoma. Sex, socioeconomic status and age at the time of diagnosis (as a continuous variable) were incorporated into the statistical model as confounders. ## Cohort study (patients with melanoma only) (stage two) In this stage of the study only those with a diagnosis of melanoma were included [fig_ref] Fig 1: Study design [/fig_ref]. Overall survival was calculated as the time from melanoma diagnosis to the time of death (outcome) or the end of the study (December 2018). Melanoma-specific survival was calculated as the time from melanoma diagnosis to the date of death from melanoma, or the end of the study for patients still alive (December 2018). Cases with missing variables were excluded from this aspect of the study. Kaplan-Meier curves were generated for smoking status and socioeconomic status, with curves compared using the logrank test. A Cox hazard proportional regression model was used to determine the association between smoking and mortality in the melanoma cohort. Sex, socioeconomic status, # Results Between 2000 and 2015, 9636 patients were diagnosed with melanoma in Wales. Stage one: case-control study Patient demographics and clinical characteristics of the cases and controls are outlined in. Data relating to smoking status were available for 7124 (73Á9%) of the melanoma cohort; 1460 current smokers (20Á5%), 3065 (43Á0%) exsmokers and 2599 (36Á5%) nonsmokers. ## Smoking After adjusting for sex, age and socioeconomic status, current smokers had 30% reduced odds for developing melanoma compared with nonsmokers, (OR 0Á70, 95% CI 0Á65-0Á76) [fig_ref] Table 3: Univariable logistic regression assessing risk factors for melanoma [/fig_ref]. There was no association between being an exsmoker or nonsmokers and melanoma (OR 1Á05, 95% CI 0Á98-1Á12). ## Socioeconomic status We observed an inverse relationship between socioeconomic status and melanoma, whereby patients from higher socioeconomic WIMD quintiles were more likely to develop melanoma. Those in the highest socioeconomic quintile (WIMD 5) were 1Á58 times more likely to develop melanoma as opposed to the lowest (HR 1Á58, 95% CI 1Á44-1Á73) [fig_ref] Table 3: Univariable logistic regression assessing risk factors for melanoma [/fig_ref]. ## Stage two: survival analysis of the melanoma cohort Demographic data [fig_ref] Table 4: Demographic characteristics of the melanoma cohort [/fig_ref] displays the demographics of the melanoma cohort. The median age at diagnosis was higher in nonsmokers (66Á7 years) and ex-smokers (64Á5 years) than in current smokers (62Á4 years). Socioeconomic status had significant variation among groups, with the current and ex-smokers being more likely to have lower socioeconomic status WIMD quintiles. Stage at diagnosis was not significantly different between smoking groups or socioeconomic status. No differences between the mean Charlson Comorbidity Index scores were noted between the smoking groups or between WIMD quintiles [fig_ref] Table 4: Demographic characteristics of the melanoma cohort [/fig_ref]. ## Mortality A total of 3103 (32Á2%) patients with melanoma died during the study period. Of these, 1688 (54Á4%) died from melanoma (melanoma listed as the primary cause of death on their death certificate) and 1415 (45Á6%) deaths were unrelated to melanoma. For patients who died from any cause, median time to death was 2Á36 years. For patients who died of melanoma, median time to death was 1Á73 years. # Univariate survival analysis Median follow-up duration of the entire cohort was 5Á22 years (range: 0-18 years). Overall survival rates were different across the three smoking status groups, with ex-smokers having lower survival than current or nonsmokers (P ≤ 0Á001). In contrast, no difference was observed across the three smoking status groups for disease-specific mortality (P = 0Á88). Overall and melanoma-specific survival rates by smoking status are shown in [fig_ref] Table 3: Univariable logistic regression assessing risk factors for melanoma [/fig_ref] (see Supporting Information). [fig_ref] Fig 3: Disease-specific survival rates by smoking status [/fig_ref] shows the overall and disease-specific survival curves, respectively, by smoking status. Overall and diseasespecific survival rates differed significantly across the WIMD quintiles [fig_ref] Table 4: Demographic characteristics of the melanoma cohort [/fig_ref] ; see Supporting Information). Figures 4 and 5 show the overall and disease-specific survival curves, respectively, by socioeconomic status. # Multivariable survival analysis After adjusting for the aforementioned factors, current smokers had an increased overall risk of death as compared with nonsmokers (HR 1Á30, 95% CI 1Á09-1Á55) [fig_ref] Table 5: Cox model for overall and disease-specific survival [/fig_ref]. There was no association between current smoking and melanomaspecific mortality. Increased odds of survival was noted in the highest socioeconomic WIMD quintile (quintile 5), compared with the lowest (quintile 1) (HR 0Á67, 95% CI 0Á56-0Á81). A similar trend was observed with disease-specific mortality (HR 0Á69, 95% CI 0Á53-0Á90). Men had an increased risk of overall and melanoma-specific death compared with women (overall HR 1Á28, 95% CI 1Á13-1Á46; disease-specific HR 1Á35, 95% CI 1Á12-1Á62). Tumour location was an important predictor of survival. For overall survival, tumours located on the upper limb were associated with increased survival compared with those on the trunk (HR 0Á73, 95% CI 0Á61-0Á88), with no association between tumours on the head and neck, and lower limbs. With regards to melanoma-specific mortality, tumours located on the trunk were associated with an increased risk of mortality when compared with those in other locations. Age was associated with a small increased risk of overall and melanoma-specific mortality (overall HR 1Á06, 95% CI 1Á05-1Á06, P ≤ 0Á001; disease-specific HR 1Á02, 95% CI 1Á01-1Á03, P ≤ 0Á001). Melanoma morphology was not associated with overall survival, however, melanoma-specific mortality was increased in those with nodular melanoma (HR 1Á23, 95% CI 0Á98-1Á54) whereas those with lentigo maligna melanoma had improved survival (HR 0Á43, 95% CI 0Á21-0Á89). The Charlson Comorbidity Index was not association with overall (HR 1Á01, 95% CI 1Á00-1Á02) or melanoma-specific survival (HR 1Á00, 95% CI 0Á99-1Á02). # Discussion We found that smokers were less likely to develop melanoma in this population-based, case-control study, but that their overall survival was reduced. After controlling for age, sex, socioeconomic status, tumour location, morphology and stage, the smoking group had an increased risk of death from all causes as compared with the nonsmoking group. However, when investigating melanoma-specific mortality, no association was observed. The mechanism responsible for the observed protective association of smoking on the risk of developing melanoma is not yet known, but several plausible hypotheses exist. Some authors hypothesize that the accumulation of nicotine in cells containing melanin suppresses the inflammatory response to ultraviolet B. [bib_ref] Melanin and nicotine: a review of the literature, Yerger [/bib_ref] [bib_ref] Transdermal nicotine suppresses cutaneous inflammation, Mills [/bib_ref] [bib_ref] Altered inflammatory responses in smokers, Mills [/bib_ref] Additionally, as smoking increases elastosis, it has been hypothesized that elastosis formation is protective of melanoma. [bib_ref] Skin aging from ultraviolet irradiance and smoking reduces risk of melanoma: epidemiological..., Grant [/bib_ref] Alternative explanations include earlier deaths in current and ex-smokers leading to survival bias, whereby those exposed to smoking die before being at risk of developing melanoma. Melanoma is not the only condition where smoking has been shown to have a favourable association, others include Parkinson disease and ulcerative colitis. [bib_ref] A meta-analysis of coffee drinking, cigarette smoking, and the risk of Parkinson's..., Hernan [/bib_ref] [bib_ref] Role of smoking in inflammatory bowel disease: implications for therapy, Thomas [/bib_ref] The protective association in Parkinson disease has been attributed to nicotine's ability to prevent brain damage and dopamine depletion. The depletion of dopamine occurs in the substantia nigra, an area of the brain populated by melanocytes. It is therefore plausible that Parkinson disease and melanoma share similar pathogenesis. [bib_ref] The association between Parkinson's disease and melanoma, Pan [/bib_ref] Numerous studies have demonstrated an increased risk of melanoma in patients with Parkinson disease and vice versa. [bib_ref] The association between Parkinson's disease and melanoma: a systematic review and metaanalysis, Huang [/bib_ref] The inverse association of smoking and the risk of developing ulcerative colitis is well reported in the literature; however, the pathogenesis is less well understood. [bib_ref] Smoking in inflammatory bowel disease: impact on disease course and insights into..., Parkes [/bib_ref] The relationship with smoking status has been investigated for nonmelanoma skin cancers. In a prospective cohort study of over one million participants, current smokers were found to have a reduced risk of developing basal cell carcinoma (BCC). Similar to our study, this 'protective' association was not observed in ex-smokers. Squamous cell carcinoma (SCC) is conversely more common in smokers. [bib_ref] Heterogeneous relationships of squamous and basal cell carcinomas of the skin with..., Pirie [/bib_ref] The Notch pathway, which functions broadly in specifying cell fates during embryogenesis and adult life, has a key role in linking the control of epidermal differentiation and proliferation. [bib_ref] Emerging role of Notch signaling in epidermal differentiation and skin cancer, Panelos [/bib_ref] Aberrant Notch signalling leads to skin cancer, although with different associations with different skin cancer types. [bib_ref] Emerging role of Notch signaling in epidermal differentiation and skin cancer, Panelos [/bib_ref] For melanoma, nodular and superficial BCC, Merkel carcinoma and SCC in sun-protected sites increased Notched signalling has an oncogenic effect. However, for basosquamous BCC and SCC on sun-exposed sites increased signalling has an oncosuppressive effect. The Notch pathway has been found to be downregulated in smokers, which could provide a further explanation on the protective association of smoking on [formula] Reference Q2 0Á09 1Á09 (0Á97-1Á20) Q3 ≤ 0Á001 1Á20 (1Á09-1Á32) Q4 [/formula] ≤ 0Á001 1Á30 (1Á18-1Á43) Q5 (highest socioeconomic status) melanoma and nodular BCC and the higher risk of SCC on sun-exposed sites. [bib_ref] Emerging role of Notch signaling in epidermal differentiation and skin cancer, Panelos [/bib_ref] [bib_ref] Activation of Notch1 signaling is required for b-catenin-mediated human primary melanoma progression, Balint [/bib_ref] [bib_ref] Growth and progression of melanoma and non-melanoma skin cancers regulated by ubiquitination, Nakayama [/bib_ref] [bib_ref] Does Notch play a tumor suppressor role across diverse squamous cell carcinomas, Zhang [/bib_ref] Although we observed that smokers appeared to be at reduced risk of melanoma, their overall survival was reduced. This finding is not surprising given the strong relationship between smoking and other life-limiting conditions, such as the majority of cancers and cardiorespiratory disease. However, consistent with the potential protective influence of smoking on melanoma development, the risk of death from melanoma was not different between the smokers and nonsmokers after adjusting for age, sex, stage of disease, morphology, socioeconomic status and tumour location. This might imply that smoking does not affect the disease progression of melanoma. This is however, not consistent with the work of Jones et al., who identified that at presentation, smokers had an increased risk of lymph node metastasis. [bib_ref] The impact of smoking on sentinel node metastasis of primary cutaneous melanoma, Jones [/bib_ref] The discrepancy may be explained by the fact that their study did not control for socioeconomic status. In addition, Jones et al. reported an association between smoking status and Breslow thickness at presentation. In our study we did not have data on Breslow thickness; however, smoking status was not associated with stage at presentation. Consistent with the published literature we found that the risk of developing melanoma was positively associated with socioeconomic status in our study. [bib_ref] Socioeconomic and lifestyle factors and melanoma: a systematic review, Jiang [/bib_ref] The underlying explanation is poorly understood and likely to be complex and multifactorial. Socioeconomic status is closely linked with lifestyle factors such as travel, sunbed use and hobbies that are also associated with sunlight exposure, with the literature supporting the notion that those that are more affluent have greater exposure to lifestyle factors that increase melanoma incidence. [bib_ref] Socioeconomic and lifestyle factors and melanoma: a systematic review, Jiang [/bib_ref] [bib_ref] Socioeconomic status and cutaneous malignant melanoma in Northern Europe, Idorn [/bib_ref] Our study also demonstrated that those in the highest socioeconomic status group were less likely to smoke. Despite higher socioeconomic status being associated with an increased risk of melanoma development, lower socioeconomic status is associated with poorer survival once diagnosed. This relationship was observed in both overall and disease-specific survival rates. This is consistent with the broader health literature where it has been shown that lower socioeconomic status is associated with premature mortality from a number of conditions such as cardiovascular disease, respiratory disease and some malignancies. [bib_ref] Mortality by deprivation and cause of death in England and Wales, Romeri [/bib_ref] In previous studies, low socioeconomic status has been associated with later stage of melanoma diagnosis; however, this was not observed in this study. Our results may be explained by the measure used to classify socioeconomic status, the WIMD score. One of the seven domains used to determine the WIMD quintile is health, which is determined by the number of limiting longterm illnesses, the all-cause death rate, cancer incidence and birthweight. Patients within the low socioeconomic status group may therefore have other attributable factors influencing survival. Limitations of this study included missing data, the lack of information available on ethnicity and ultraviolet light exposure. As with any population-based study, missing data prevented analysis on the total cohort. Data were missing for some of the cohort on smoking status and stage of disease. Smoking status was obtained from the WLGP, as recorded during patient's consultations with their general practitioner. To date, the WLGP covers 80% of general practices across Wales. Of the 2512 patients for whom smoking data were absent, 2431 (96Á8%) belonged to general practices not contributing data to the SAIL Databank. It is therefore assumed that data for this variable were missing at random and would not bias the results. Additionally, information was not available on the quantity of tobacco smoked by participants. The Read codes listed in the(see Supporting Information) do capture some information on the amount of smoking. In practice, these codes were rarely utilized by general practitioners, with the majority simply recording 137R (current smoker) and therefore we were unable to provide meaningful results. This is a substantial limitation as the cumulative exposure to tobacco was not assessed, thus it was not possible to calculate a doseresponse relationship. When stage of melanoma was not recorded in the WCISU data, and could not be obtained from other linked data, these data were missing. To assess the effect of this missingness, a sensitivity analysis was performed. Missing data were incorporated into the regression model as a separate category for stage. This was found not to affect the statistical significances outlined in the results section. A further limitation of population-based studies using routinely collected data is incomplete control of confounding, that of data that are not specified, incompletely captured or misclassified, namely tumour location (relating to ICD-10 Code C43Á9 melanoma unspecified) and tumour morphology [M7203 -MM NOS (melanomanot otherwise specified)]. The classification codes used to extract smoking status from general practitioner data have shown to classify 8Á6% ex-smokers as never smokers. Any misclassification would not significantly bias the results. Ethnicity is only available on special request within the SAIL Databank and was therefore not incorporated into the statistical model. In Wales, population statistics reveal that 95% of the population are white and therefore the significance of ethnicity on the results would be minimal.In conclusion, this is the largest study to date indicating that smoking has an inverse relationship on the risk of developing melanoma. While the detrimental repercussions of smoking are well documented, further work is required to uncover the mechanism underlying this relationship, including further assessment about survival bias. If a biological association seems likely, this could lead to the development of novel prevention and treatment options, opening up a new wave of medical therapy for melanoma. Furthermore, this work reinforces the ongoing association between melanoma and socioeconomic status. Despite numerous public health strategies, higher socioeconomic groups continue to have a higher incidence of melanoma, however, lower socioeconomic status is related to poor survival once melanoma is diagnosed. The implications of these results, in a country such as the U.K. where health care is free to all, are significant. Further work is required to investigate the barriers to care that may exist for the lowest socioeconomic status group so that policies can be implemented to prevent healthcare inequality and improve melanoma outcomes for all. ## Supporting information Additional Supporting Information may be found in the online version of this article at the publisher's website: [fig_ref] Table 1: List of databases used and their description Database Description Annual District Death... [/fig_ref] The Reporting of studies Conducted using Observational Routinely collected health Data (RECORD) statement.List of Read codes used for smoking status in this study. [fig_ref] Table 3: Univariable logistic regression assessing risk factors for melanoma [/fig_ref] Overall and disease-specific survival rates by smoking status. [fig_ref] Table 4: Demographic characteristics of the melanoma cohort [/fig_ref] Overall and disease-specific survival rates by socioeconomic status. [fig] Fig 1: Study design. SAIL, Secure Anonymised Information Linkage; WCISU, Welsh Cancer Intelligence and Surveillance Unit; WDS, Welsh Demographic Service; WIMD, Welsh Index of Multiple Deprivation. [/fig] [fig] Fig 3: Disease-specific survival rates by smoking status. [/fig] [fig] Fig 4 Fig 5: Overall survival by socioeconomic status. WIMD, Welsh Index of Multiple Deprivation. Disease-specific survival by socioeconomic status. WIMD, Welsh Index of Multiple Deprivation. [/fig] [table] Table 1: List of databases used and their description Database Description Annual District Death Extract Collected from the Office for National Statistics, containing death registration information, relating to Welsh residents including those who died outside of Wales. Outpatient Dataset for Wales Administrative and clinical data obtained from outpatient appointments in Wales. Patient Episode Database for Wales Administrative and clinical data for all hospital admissions, including diagnosis and operations performed. Welsh Cancer Intelligence and Surveillance Unit The national cancer registry for Wales. Captures all Welsh patients with melanoma from a number of sources: multidisciplinary team data, pathology data, other routine data sources in Wales and the English cancer registry. Welsh Longitudinal General Practice Administrative and clinical data from all patient visits to a general practitioner. Welsh Demographic Service Dataset Administrative data about individuals resident or registered in Wales that have used National Health Service services.melanoma stage at diagnosis and age at diagnosis as a continuous variable were incorporated into the model as confounders. Both overall survival (deaths from any cause) and melanomaspecific survival (defined on their death registration held within the Annual District Death Extract) were analysed in the melanoma cohort. All data were analysed using IBM SPSS Statistics for Windows (Released 2017, version 25Á0, IBM Corp, Armonk, NY, U.S.A.). Statistical significance was assumed with a P < 0Á05. [/table] [table] Table 3: Univariable logistic regression assessing risk factors for melanoma [/table] [table] Table 4: Demographic characteristics of the melanoma cohort [/table] [table] Table 5: Cox model for overall and disease-specific survival [/table]

Metabolic disorders and the risk of head and neck cancer: a protocol for a systematic review and meta-analysis # Introduction Head and neck cancer squamous cell carcinoma (HNSCC), which includes cancer of the oral cavity, oropharynx, hypopharynx and larynx, is the world's sixth most common cancer, with the highest incidence in men and those over 70 years old.Established risk factors include smoking, alcohol and the human papillomavirus (HPV), which has mainly been linked to oropharyngeal cancer. [bib_ref] The changing aetiology of head and neck squamous cell cancer: a tale..., Thomas [/bib_ref] Despite the significant reduction in smoking,and a fall in alcohol consumption, 4 in the UK over the previous two decades, incidence rates of head and neck cancers have continued to rise by around a third.Given this potentially changing aetiology, 2 further exploration of less well known risk factors is warranted to target prevention and identify patients with early disease. People living with obesity and associated conditions, such as type 2 diabetes (T2D), have an increased risk of developing certain cancers (eg, liver and pancreas), but the epidemiological evidence surrounding HNSCC is not conclusive. Recent studies have demonstrated elevated glucose levels before or at the time of diagnosis, which may play a role in the pathogenesis and progression of cancer. [bib_ref] The impact of diabetes in head and neck cancer, Vilaseca [/bib_ref] Carcinogenesis in HNSCC is driven by diverse signalling pathways; however, little is known about the role of metabolism, [bib_ref] Anti-Tumor effects of metformin on head and neck carcinoma cell lines: a..., Rêgo [/bib_ref] despite metabolic reprogramming being a recognised hallmark of cancer. 9 ## Strengths and limitations of this study ⇒ This systematic review will be the first to comprehensively review the literature and provides a metaanalysis of the effect of metabolic disorders and the risk of incident head and neck cancer squamous cell carcinoma (HNSCC). ⇒ The publication of this protocol provides a clear representation of the methods used in this review for transparency and to prevent future duplication. ⇒ This systematic review will be one of the first to use the Risk Of Bias In Non-randomised Studies-of Exposures tool. ⇒ Despite the metabolic disorders being described as separate entities, the authors recognise the potential for disease processes to be related. ⇒ Among cohort studies, there is significant variability in terms of length of follow-up for the outcome of interest, HNSCC. ## Open access T2D is a common condition characterised by insulin resistance and hyperglycaemia, which results in wholebody metabolic dysregulation. [bib_ref] Discovery of biomarkers for glycaemic deterioration before and after the onset of..., Koivula [/bib_ref] In 2015, the estimated worldwide diabetes prevalence for adults was predicted to rise to 642 million by 2040. [bib_ref] IDF diabetes atlas: global estimates for the prevalence of diabetes for 2015..., Ogurtsova [/bib_ref] In addition, public health strategies have not successfully addressed the current 'obesity pandemic'. [bib_ref] Current estimates of the economic cost of obesity in the United States, Wolf [/bib_ref] [bib_ref] Long term maintenance of weight loss with non-surgical interventions in obese adults:..., Dombrowski [/bib_ref] [bib_ref] A systematic review and mixed treatment comparison of pharmacological interventions for the..., Gray [/bib_ref] [bib_ref] Long-Term drug treatment for obesity: a systematic and clinical review, Yanovski [/bib_ref] [bib_ref] Body mass index: has epidemiology started to break down causal contributions to..., Corbin [/bib_ref] One large pooled analysis demonstrated a weak positive association between T2D and HNSCC (OR 1.09; 95% CI: 0.95 to 1.24), which was stronger among those who never smoked cigarettes (OR 1.59; 95% CI: 1.22 to 2.07). [bib_ref] History of diabetes and risk of head and neck cancer: a pooled..., Stott-Miller [/bib_ref] While obesity is a potent risk factor for T2D, the relationship between body mass index (BMI) and HNSCC is not straightforward, with both positive and inverse associations observed. [bib_ref] Body mass index and risk of subtypes of head-neck cancer: the Netherlands..., Maasland [/bib_ref] Another study found that a positive association with BMI was only observed in never smokers. [bib_ref] Anthropometry and head and neck cancer:a pooled analysis of cohort data, Gaudet [/bib_ref] In the largest study to date, waist circumference and waist-to-hip ratio were positively associated with higher HNSCC risk, regardless of smoking status. [bib_ref] Anthropometry and head and neck cancer:a pooled analysis of cohort data, Gaudet [/bib_ref] Metabolic syndrome has been described as a clustering of disorders including obesity, hypertension, hyperglycaemia and dyslipidaemia. A moderate inverse association has been observed between metabolic syndrome and HNSCC (OR 0.81; 95% CI: 0.78 to 0.85), but again these results were modified by tobacco use. [bib_ref] Type II diabetes and metabolic syndrome in relation to head and neck..., Stott-Miller [/bib_ref] Treatment for these conditions using medications such as statins, 20 or metformin, 21 may also reduce the risk of developing HNSCC. Given the growing pandemic of metabolic disorders, understanding how these alterations affect the risk of carcinogenesis may identify those who are high risk. This could drive targeted prevention, earlier detection and perhaps the identification or repurposing of drug targets for treatment in HNSCC. [bib_ref] Hyperglycemia associated metabolic and molecular alterations in cancer risk, progression, treatment, and..., Ramteke [/bib_ref] ## Rationale The complex metabolic changes associated with T2D, obesity, dyslipidaemia and hypertension may alter the risk of certain cancers, but the evidence for head and neck cancer is inconclusive. This study aims to identify, collate and synthesise all relevant studies including adult participants, to determine whether the risk of developing incident HNSCC is influenced by T2D, obesity, dyslipidaemia and hypertension, using relevant effect measures (eg, OR or risk ratios (RR)). Where available, we will further stratify by subsite (ie, oral cavity, oropharynx, hypopharynx and larynx), as well as by HPV status. # Methods ## Research question Do metabolic disorders affect the risk of developing head and neck cancer? ## Study design Randomised controlled trials are informative for assessing causal effects; however, due to the nature of the exposures in this study, inclusion of this design is not feasible. Therefore, we will focus on observational (eg, cohort studies) reporting the risk of metabolic disorders on incident HNSCC. ## Eligibility criteria Population: participants over 18 years old, of either sex, from any ethnic background. Exposures (1) T2D, (2) obesity, (3) dyslipidaemia and (4) hypertension. These will be collectively described as 'metabolic disorders'. Comparison: participants who have not been diagnosed with the afore-mentioned metabolic disorders. Outcome: incident HNSCC. The study will report in line with the Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) statement and has been pre-registered on International Prospective Register of Systematic Reviews (PROSPERO) in 2021. ## Search strategy A systematic search strategy (online supplemental table 1) has been formulated by clinicians, scientific researchers and a specialist librarian for surgery, head and neck and medicine. Medical Subject Headings and keywords will be iteratively combined with the Boolean operators AND, OR and NOT. Multiple databases will be searched from January 1966 to November 2021, including OVID SP versions of Medline and EMBASE, using the formulated search strategy (online supplemental table 1). Preprint servers including medRxiv and bioRxiv will also be searched. In addition, the following electronic bibliographic databases will be searched: Cochrane Library, EThOS, Google Scholar, Open Grey and Clinical-Trials. gov, to identify articles from the grey literature and conference proceedings using a modified search strategy (online supplemental table 2). References extracted from the full-length articles will be reviewed to identify other publications of interest. Duplicate articles will be removed using Covidence. 23 ## Data management The results from searches will be imported into Covidence software, to improve reference management and workflow. This will also populate a PRISMA flow diagram. [bib_ref] The PRISMA statement for reporting systematic reviews and meta-analyses of studies that..., Liberati [/bib_ref] Data screening and extraction All titles and abstracts will be screened by two authors AG and CR, with conflicts resolved by MG. Screening records and decisions will be kept in Covidence. Data will be extracted from titles that meet the inclusion criteria (table 1). The data extraction form (Online supplemental table 3) will be piloted using five included titles with AG, CR and MG independently completing data extraction. If amendments are necessary, they will be performed at this point, with consensus approval required prior to final use of the form. Subsequent to this, CR and MG will double data extract independently for each title, any disagreements will be discussed among AG, CR and MG with consensus approval required. Exposure definitions 1. The WHO defines T2D as a chronic disease that occurs when the body cannot effectively use the insulin it produces and is largely the result of excess body weight and physical inactivity. A diagnosis of T2D was defined as symptoms such as polyuria or polydipsia, plus : -A random blood plasma glucose concentra-tion≥11.1 mmol/L. -A fasting plasma glucose concentration≥7.0 mmol/L (whole blood≥6.1 mmol/L). -Two-hour plasma glucose concentra-tion≥11.1 mmol/L, two hours after 75 g anhydrous glucose in an oral glucose tolerance test. -Glycated haemoglobin (HbA1c) 6.5% or more (48 mmol/mol and above). Relevant International Classification of Disease (ICD) codes used to define T2D will also be acceptable. 2. The WHO definition ofBMI of 30 or above must have been used to define obesity. 27 BMI if calculated from self-reported height and weight or taken from medical records or measured at baseline will be acceptable. 3. Dyslipidaemia was classified as serum total cholesterol, low-density lipoprotein cholesterol, triglycerides, apolipoprotein B or lipoprotein(a) concentrations above the 90th percentile, or high-density lipoprotein cholesterol or apolipoprotein concentrations below the 10th percentile for the general population.Relevant ICD codes used to define dyslipidaemia will also be acceptable. 4. The WHO definition of hypertension as: -A systolic blood pressure reading of ≥140 mm Hg. -A diastolic blood pressure readings on both days of ≥90 mm Hg, must have been used. Relevant ICD codes used to define hypertension will also be acceptable. ## Outcome definitions All diagnoses of incident HNSCC included in this study should have been confirmed by histology by a trained pathologist. Acceptable cancer diagnosis may be reported using ICD codes.Cancer cases for the disease of interest have the following ICD-10 codes: oral cavity (C02.0-C02.9, C03.0-C03.9, C04.0-C04.9, C05.0-C06.9) oropharynx (C01.9, C02.4, C09.0-C10.9), hypopharynx (C13.0-C13.9), overlapping (C14 and combination of other sites) and 25 cases with unknown ICD code (other). Older versions of ICD code, for example, ICD-8 and ICD-9 were also accepted. With respect to HPV diagnosis, only high-risk subtypes HPV16, , and 33 will be included. These are should be detected using HPV-DNA via PCR and/or in situ hybridisation examination, usually used in combination with immunohistochemistry for p16. ## Data analysis and synthesis The analysis and synthesis process will be performed in two phases. The first phase consists of estimating the effect of each metabolic disorder (T2D, obesity, dyslipidaemia and hypertension) on incident HNSCC separately for each included study. Effects should be reported as HR, standardised incidence ratio, OR or RR, with 95% CIs or in a format that will enable us to calculate these. The second phase will focus on estimating the combined effect across studies using meta-analysis, using metan software in Stata (Stata Statistical Software: Release V. [bib_ref] Body mass index: has epidemiology started to break down causal contributions to..., Corbin [/bib_ref]. College Station, Texas, USA: StataCorp LLC). Summary effects with their corresponding 95% CIs will be derived with the method of DerSimonian and Laird. [bib_ref] Meta-Analysis in clinical trials, Dersimonian [/bib_ref] This will be performed separately for each metabolic disorder. The analysis will yield information about the heterogeneity of effects across studies, using Cochran's Q test and Higgins' I 2 statistic. Due to the heterogeneity in study Study designs such as case reports or case series. In addition, cross-sectional, case-control and narrative review studies will be excluded due to the inability to infer a temporal relationship. Studies must report an OR or risk ratio, or data which will allow these to be calculated. Human studies only, no in vivo animal or in vitro cell line studies. Exposures: type 2 diabetes, obesity, dyslipidemia and hypertension with definitions as described in the study protocol. Studies of head and neck epithelial dysplasia, potentially malignant disease or carcinoma in situ. An outcome of head and neck cancer diagnosis which may be human papilloma virus (HPV) positive or negative. High risk types HPV16, 18, 31 and 33 only will be included. Squamous cell carcinoma of other sites, for example, nasopharynx, salivary gland, oesophagus, skin or lung due to differing aetiology, histological subtypes and risk factors, for example, Epstein-Barr virus. Open access design, exposure definition and outcome reporting in the included studies, meta-analysis may not be reasonable. If appropriate, to further explore the sources of heterogeneity if present, meta-regression analyses will be performed according to age, geographic location, year of publication, methods of exposure assessment, risk of bias score, length of follow-up, the number of cases and adjustment for confounding factors including smoking and alcohol use. Small study effects, which may reflect publication bias, study quality or other sources of heterogeneity, will be assessed using funnel plots and Egger's regression asymmetry tests. ## Subgroup analyses Where possible, we will stratify by oral, oropharyngeal, hypopharyngeal and laryngeal subsite and by HPV status as post-hoc analyses, to determine if effects seen are specific to a particular anatomical region or associated with specific tumour types. ## Risk of bias in studies Two authors will independently extract the relevant risk factor information during data extraction using a preliminary version of the Risk of Bias in Non-randomised Studies-of Exposures (ROBINS-E) tool (https://www. bristol.ac.uk/population-health-sciences/centres/ cresyda/barr/riskofbias/robins-e/).The ROBINS-E tool aims to assess the result of interest from an observational study for risk of bias, where that study is designed to assess the effect of exposure on outcome.Disagreements will be recorded and discussed with a third author for resolution. This assessment will be used in evaluating the strength of evidence from the included studies. ## Amendments Any amendments to the initial protocol will be included in an update to the PROSPERO record, including information on amendment type, reasoning and a timestamp. Deviation from this protocol will be described in the full systematic review paper. ## Dissemination This systematic review will be published in a relevant peer-reviewed journal and findings from this work may be presented at scientific meetings (both poster and oral presentations). Competing interests None declared. Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research. ## Patient consent for publication not applicable. Provenance and peer review Not commissioned; externally peer reviewed. Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise. Open access This is an open access article distributed in accordance with the Creative Commons Attribution 4.0 Unported (CC BY 4.0) license, which permits others to copy, redistribute, remix, transform and build upon this work for any purpose, provided the original work is properly cited, a link to the licence is given, and indication of whether changes were made. See: https://creativecommons.org/ licenses/by/4.0/. [table] Table 1: Study selection criteriaAll studies published from January 1966 in the English language. The study is not based on incident head and neck squamous cell carcinoma, or contains only prevalent data, meaning a temporal relationship cannot be inferred. [/table]

Resistance to organic hydroperoxides requires ohr and ohrR genes in Sinorhizobium meliloti Background: Sinorhizobium meliloti is a symbiotic nitrogen-fixing bacterium that elicits nodules on roots of host plants Medicago sativa. During nodule formation bacteria have to withstand oxygen radicals produced by the plant. Resistance to H 2 O 2 and superoxides has been extensively studied in S. meliloti. In contrast resistance to organic peroxides has not been investigated while S. meliloti genome encodes putative organic peroxidases. Organic peroxides are produced by plants and are highly toxic. The resistance to these oxygen radicals has been studied in various bacteria but never in plant nodulating bacteria. Results: In this study we report the characterisation of organic hydroperoxide resistance gene ohr and its regulator ohrR in S. meliloti. The inactivation of ohr affects resistance to cumene and ter-butyl hydroperoxides but not to hydrogen peroxide or menadione in vitro. The expression of ohr and ohrR genes is specifically induced by organic peroxides. OhrR binds to the intergenic region between the divergent genes ohr and ohrR. Two binding sites were characterised. Binding to the operator is prevented by OhrR oxidation that promotes OhrR dimerisation. The inactivation of ohr did not affect symbiosis and nitrogen fixation, suggesting that redundant enzymatic activity exists in this strain. Both ohr and ohrR are expressed in nodules suggesting that they play a role during nitrogen fixation.Conclusions: This report demonstrates the significant role Ohr and OhrR proteins play in bacterial stress resistance against organic peroxides in S. meliloti. The ohr and ohrR genes are expressed in nodule-inhabiting bacteroids suggesting a role during nodulation. # Background Sinorhizobium meliloti is a soil bacterium that must survive and proliferate in various adverse conditions. S. meliloti is also able to establish a symbiotic partnership with Medicago sativa leading to the formation of nodules. In nodules, the bacterium differentiates in bacteroids and fixes atmospheric nitrogen. Within the soil and during nodulation, S. meliloti copes with various stresses imposed by the environmentor by plant responses to bacterial invasion [bib_ref] Reactive oxygen and nitrogen species and glutathione: key players in the legume-Rhizobium..., Pauly [/bib_ref] [bib_ref] Responses of rhizobia to desiccation in relation to osmotic stress, oxygen, and..., Vriezen [/bib_ref]. While nodulation is a close association between plant and S. meliloti, bacteria are initially recognised as intruders and induce an oxidative burst [bib_ref] Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction, Santos [/bib_ref]. An increased production of reactive oxygen species (ROS), including superoxides, H 2 O 2 and organic hydroperoxides is an important component of plant defences [bib_ref] Role of active oxygen species and NO in plant defence responses, Bolwell [/bib_ref]. In free living bacteria oxidative stress results from aerobic metabolism [bib_ref] Metabolic sources of hydrogen peroxide in aerobically growing Escherichia coli, Gonzalez-Flecha [/bib_ref] , ROS are produced as a result of the incomplete reduction of molecular oxygen [bib_ref] Pathways of oxidative damage, Imlay [/bib_ref]. Hence, both in free living and symbiotic stages, S. meliloti produces enzymes to detoxify ROS. Only those that detoxify superoxide anion and H 2 O 2 have been studied extensively Superoxides are detoxified by two superoxide dismutases [bib_ref] Sinorhizobium meliloti orpE2 is necessary for H 2 O 2 stress resistance..., Flechard [/bib_ref] [bib_ref] Critical protective role of bacterial superoxide dismutase in rhizobium-legume symbiosis, Santos [/bib_ref] , H 2 O 2 by three catalases (KatA, KatB and KatC) [bib_ref] Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago sativa symbiosis and..., Jamet [/bib_ref] and a chloroperoxidase (Cpo) [bib_ref] Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021, Barloy-Hubler [/bib_ref]. Little is known about resistance to organic peroxides (OHPs) in S. meliloti. OHPs are generated as part of the active defence response of plants [bib_ref] Active oxygen in plant pathogenesis, Baker [/bib_ref] [bib_ref] Lipid peroxidation in cotton, Xanthomonas interactions and the role of lipoxygenases during..., Jalloul [/bib_ref]. OHPs are highly toxic. They participate in free radical reactions that generate more toxic ROS by reacting with membranes and other macromolecules [bib_ref] Oxygen toxicity, oxygen radicals, transition metals and disease, Halliwell [/bib_ref]. Thus, detoxification of OHPs is important for bacterial survival and proliferation. Bacteria possess two systems to protect themselves against organic peroxide toxicity. Peroxiredoxines have been shown to be the main peroxide detoxification enzymes in eukaryotes and bacteria [bib_ref] Peroxiredoxins in bacterial antioxidant defense, Dubbs [/bib_ref] [bib_ref] Peroxiredoxins: a historical overview and speculative preview of novel mechanisms and emerging..., Rhee [/bib_ref]. Alkyl hydroperoxidase reductase (Ahp) constitutes the best characterised member of peroxiredoxin family [bib_ref] Amphibacillus xylanus NADH oxidase and Salmonella typhimurium alkyl-hydroperoxide reductase flavoprotein components show..., Niimura [/bib_ref] [bib_ref] Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein..., Poole [/bib_ref]. This enzyme is composed of a reductase subunit and a catalytic subunit reducing organic peroxides to alcohols [bib_ref] Bacterial defenses against oxidants: mechanistic features of cysteine-based peroxidases and their flavoprotein..., Poole [/bib_ref]. The second class of OHP detoxification enzymes (OsmC/Ohr family) is only found in bacteria [bib_ref] Bacterial Ohr and OsmC paralogues define two protein families with distinct functions..., Atichartpongkul [/bib_ref]. The Ohr (Organic Hydroperoxide Resistance) protein first discovered in Xanthomonas campestris [bib_ref] Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with..., Mongkolsuk [/bib_ref] , and OsmC (Osmotically inducible protein) [bib_ref] Osmotic induction of gene osmC expression in Escherichia coli K12, Gutierrez [/bib_ref] are hydroperoxide peroxidases catalysing the reduction of hydroperoxides into their corresponding alcohols [bib_ref] Organic hydroperoxide resistance gene encodes a thiol-dependent peroxidase, Cussiol [/bib_ref] [bib_ref] Structural and functional features of the Escherichia coli hydroperoxide resistance protein OsmC, Lesniak [/bib_ref]. Both Ohr and OsmC are structurally and functionally homologous proteins. They are homodimeric with the active sites on either side of the molecule [bib_ref] Structural and functional features of the Escherichia coli hydroperoxide resistance protein OsmC, Lesniak [/bib_ref] [bib_ref] Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr, Lesniak [/bib_ref]. Their active sites contain two highly conserved cysteines which are involved in peroxide metabolism [bib_ref] Structural and functional characterization of the Pseudomonas hydroperoxide resistance protein Ohr, Lesniak [/bib_ref] [bib_ref] Crystallographic structure and biochemical analysis of the Thermus thermophilus osmotically inducible protein..., Rehse [/bib_ref]. Despite this conservation of the proteins, OsmC and Ohr display different patterns of regulation and distinct physiological functions [bib_ref] Structural and functional features of the Escherichia coli hydroperoxide resistance protein OsmC, Lesniak [/bib_ref]. The expression of ohr is specifically induced by organic peroxides and not by ethanol and osmotic stress [bib_ref] Bacterial Ohr and OsmC paralogues define two protein families with distinct functions..., Atichartpongkul [/bib_ref] , while osmC is not induced by organic peroxides; instead it is induced by ethanol and osmotic stress and controlled by multiple general stress responsive regulators [bib_ref] Peroxiredoxins in bacterial antioxidant defense, Dubbs [/bib_ref]. The inactivation of ohr, but not osmC, reduces the resistance only against organic peroxides, and not to other oxidants [bib_ref] Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with..., Mongkolsuk [/bib_ref]. The expression of ohr is regulated by the organic peroxide-inducible transcription repressor OhrR, a member of MarR family. Structural data are available for OhrR of Bacillus subtilis [bib_ref] Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of..., Hong [/bib_ref] and OhrR of X. campestris [bib_ref] Structural mechanism of organic hydroperoxide induction of the transcription regulator OhrR, Newberry [/bib_ref]. OhR functions as a dimeric repressor that binds the ohr promoter region in the absence of organic peroxides. Derepression results from the oxidation of a highly conserved active site cysteine that resides near the NH 2 terminus of the protein [bib_ref] The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic..., Fuangthong [/bib_ref]. B. subtilis OhrR contains only one cysteine; its oxidation leads to the formation of a mixed disulfide with a low molecular weight thiol, a cyclic sulfenamide, or overoxidation to sulfinic or sulfonic acids [bib_ref] Oxidation of a single active site suffices for the functional inactivation of..., Eiamphungporn [/bib_ref]. In other bacteria, like X. campestris, OhrR contains a second cysteine located on the COOH extremity of the OhrR protein (C127 for X. campestris). Oxidation of the protein initiates by the formation of a sulphenic derivative of the reactive cysteine (C22) followed by the formation of a disulfide bond with C127 of the other OhrR subunit [bib_ref] Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor..., Panmanee [/bib_ref]. While ohr homologues are widely distributed in bacterial genomes [bib_ref] Bacterial Ohr and OsmC paralogues define two protein families with distinct functions..., Atichartpongkul [/bib_ref] , the role of ohr and ohrR was only studied in a few number of bacteria: X. campestris, B. subtilis, Agrobacterium tumefasciens, Pseudomonas aeruginosa and Streptomyces coelicolor [bib_ref] Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with..., Mongkolsuk [/bib_ref] [bib_ref] Mongkolsuk S: ohrR and ohr are the primary sensor/regulator and protective genes..., Chuchue [/bib_ref] [bib_ref] Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to..., Mostertz [/bib_ref] [bib_ref] Genetic and physiological characterization of ohr, encoding a protein involved in organic..., Ochsner [/bib_ref] [bib_ref] Dual role of OhrR as a repressor and an activator in response..., Oh [/bib_ref] [bib_ref] Analyses of regulatory mechanism and physiological roles of Pseudomonas aeruginosa OhrR, a..., Atichartpongkul [/bib_ref]. In many bacteria, peroxide stress was studied only via H 2 O 2 stress. In S. meliloti, H 2 O 2 resistance has been extensively studied [bib_ref] Sinorhizobium meliloti orpE2 is necessary for H 2 O 2 stress resistance..., Flechard [/bib_ref] [bib_ref] Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago sativa symbiosis and..., Jamet [/bib_ref] [bib_ref] Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021, Barloy-Hubler [/bib_ref] while OHP resistance is poorly understood. This study aims at evaluating the role of ohr and ohrR genes on OHP resistance in S. meliloti. The analysis of the biochemical properties of ohr and ohrR mutants and the expression pattern suggests that this system should play an important role in sensing and protection of S. meliloti from OHPs. # Results ## Identification of ohr and ohrr homologues in s. meliloti Blast search of S. meliloti genome for homologues of X. campestris Ohr protein revealed two paralogues, SMa2389 and SMc00040, showing 52 and 57% identity respectively with Ohr of X. campestris. They possess conserved active site cysteines of Ohr/OsmC proteins [bib_ref] Bacterial Ohr and OsmC paralogues define two protein families with distinct functions..., Atichartpongkul [/bib_ref]. SMa2389 is annotated as OsmC. SMc00040 has been shown to be induced by peroxide stress [bib_ref] Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021, Barloy-Hubler [/bib_ref] ; it is divergently located from a gene encoding a MarR family regulator that has 49 and 45% identity with the OhrR regulatory protein of X. campestris and B. subtilis respectively. SMc01945 has been previously published as OhrR like repressor since it presents 40% identity with OhrR of X. campestris [bib_ref] Smc01944, a secreted peroxidase induced by oxidative stresses in Sinorhizobium meliloti 1021, Barloy-Hubler [/bib_ref] ; the adjacent gene cpo (SMc01944) has been shown to encode a secreted peroxidase. Co-localisation on the genome of ohr and ohrR was found in all bacteria in which these genes were investigated [bib_ref] Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with..., Mongkolsuk [/bib_ref] [bib_ref] Mongkolsuk S: ohrR and ohr are the primary sensor/regulator and protective genes..., Chuchue [/bib_ref] [bib_ref] OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant..., Fuangthong [/bib_ref] , suggesting that SMc00040 and SMc00098 encodes respectively Ohr and OhrR proteins. ohr mutant growth is inhibited by organic peroxides In order to investigate the role of ohr (SMc00040) and ohrR (SMc00098) in oxidative stress defence, S. meliloti strains with an ohrR deletion or carrying an insertion in ohr were constructed. The ability of these mutants to resist exposure to oxidants was evaluated; neither of the two had any growth defect when grown aerobically in complete medium LB or in minimal medium GAS. Moreover they possessed the same plating efficiency as wild type strain. The influence of organic peroxides on growth of wild type, ohr and ohrR strains was analysed by adding increasing amounts of t-butyl hydroperoxide (tBOOH) and cumene hydroperoxide (CuOOH) to LB medium and determining the maximal OD 570 nm reached by the cultures. A concentration of 2 mM tBOOH abolished the growth of ohr mutant while the growth yield of ohrR and parental strains was unaffected. Similarly 0.5 mM of CuOOH abolished growth of ohr strain but did not affect growth yield of ohrR and parental strains. Disk diffusion assays were used to determine if ohr and ohrR mutations affected resistance to ROS. The ohr mutant was less resistant than its parental strain when challenged with organic peroxides as shown by the zones of growth inhibition: 4.1 ± 0.2 cm for CuOOH and 3.1 ± 0.1 cm for tBOOH versus to 2.3 ± 0.2 and 2.5 ± 0.3 cm for wild type strain. In contrast, ohrR mutation did not affect the resistance of S. meliloti against tBOOH and CuOOH since inhibition zones were not significantly (p value ≤ 0.01) different from those of wild type strain . The ohr-ohrR mutant behaved identically to the ohr mutant . In other experiments, ohr and ohrR mutants were complemented by the moderate copy number plasmid pBBR1-MCS2 bearing wild type alleles of ohr (pBBohr) or ohrR (pBBohrR). The empty vector did not affect the resistance of wild type or mutants against tBOOH and CuOOH. Plasmid vector carrying ohrR + allele also did not affect the resistance to OHPs of these three strains. In contrast the introduction of pBBohr in ohr mutant dramatically improved resistance to both tBOOH and CuOOH . These results showed that Ohr is important in the defence against organic peroxides in S. meliloti. In comparison with parental strain, ohr and ohrR mutants were not significantly affected in resistance to H 2 O 2 and menadione; inhibition zones were nearly identical for the three strains. No alteration of this resistance was observed after complementation of the mutations with pBBohr or pBBohrR. This result agrees with the role of Ohr in other organisms and its specificity for organic peroxide resistance. ## Regulation of ohr and ohrr genes The transcriptional activity of ohr and ohrR genes was assayed in strain R7.16 carrying ohr::lacZ and ohrR::uidA transcriptional fusions in tandem with wild type copies of ohr and ohrR genes. The expression of these fusions was analysed in LB medium and in the minimal medium GAS. No difference was observed between both media. The expression of ohr::lacZ and ohrR::uidA was followed throughout growth till the late stationary growth phase. The expression of these two genes remained constant; no variation was observed after growth arrest. Adding NaCl to the medium during exponential growth or during stationary growth phase did not affect ohr or ohrR expression (data not shown). Such a result is expected for Ohr and clearly differentiates it from OsmC proteins that are induced by NaCl [bib_ref] Peroxiredoxins in bacterial antioxidant defense, Dubbs [/bib_ref]. The influence of peroxides was analysed by introducing into the medium a concentration of peroxide that did not affect the development of exponentially growing cells. Expression of ohr was induced 4-fold in the presence of 1.6 mM tBOOH, a 7-fold induction was observed with 0.25 mM CuOOH. The addition of 10 mM H 2 O 2 resulted in a 2-fold induction of ohr [fig_ref] Figure 2: Induction of the expression of ohr and ohrR by peroxides [/fig_ref]. Induction of ohrR was also observed when cultures were exposed to tBOOH and CuOOH, induction ratios were lower than those observed for ohr gene. In contrast H 2 O 2 did not affect ohrR expression [fig_ref] Figure 2: Induction of the expression of ohr and ohrR by peroxides [/fig_ref]. ## Ohrr regulates ohr expression A plasmid bearing ohr::lacZ transcriptional fusion (pE1541) was introduced into the ohrR mutant and the parental strain. The expression of the fusion was analysed in LB medium in the absence of organic peroxides and 1 h after 0.25 mM CuOOH addition. In the absence of peroxide, the expression of ohr::lacZ fusion was greater in the ohrR mutant than in the wild type strain (18.5 ± 1.3 and 9.6 ± 0.7 μmol of substrate hydrolysed min -1 mg of protein -1 respectively). After CuOOH addition, the expression of ohr::lacZ was similar in ohrR mutant and parental strain (16.7 ± 1.4 and 17.5 ± 1.5 μmol of substrate hydrolysed min -1 mg of protein -1 respectively). These results are in accordance with repression of ohr promoter by the OhrR regulator. ## Ohrr binds to ohr-ohrr intergenic region The binding of OhrR to ohr-ohrR intergenic region was analysed by gel mobility shift assay. In a first attempt, a 113 bp DNA fragment encompassing the entire ohr- Resistance of the ohr and ohrR mutants to ROS. The resistance of wild type (WT), ohr, ohrR, ohr-ohrR mutants and ohr mutant complemented by plasmids pBBR1-MSC2 [ohr (pBB2)] and pBBohr [ohr (pBBohr)] was analysed by disk diffusion assay on LB plates as described in methods. The data correspond to five independent experiments; standard deviation is indicated (bars). ohrR intergenic region and ended at the initiation codons of ohr and ohrR, was used as a probe [fig_ref] Figure 3: Localisation of OhrR binding sites [/fig_ref]. Two retarded bands were observed in the presence of OhrR [fig_ref] Figure 3: Localisation of OhrR binding sites [/fig_ref]. The intergenic region between SMb20903 and SMb20964 (this latter gene encoding the putative AhpC protein of S. meliloti) was used as a negative control. No specific binding of OhrR protein to this DNA fragment was observed (data not shown). We took advantage of restriction sites located within the ohr-ohrR intergenic region to define further OhrR binding site. ApoI cleaved once this fragment giving a 17 bp and a 96 bp fragment. In the presence of OhrR protein the longer fragment produced two shifted bands [fig_ref] Figure 3: Localisation of OhrR binding sites [/fig_ref]. Two HpaII sites are located within ohr-ohrR intergenic region; HpaII cleavage produced three fragments of 26, 29 and 58 bp. In the presence of OhrR, the intensity of the 58 bp fragment decreased and two retarded bands were observed [fig_ref] Figure 3: Localisation of OhrR binding sites [/fig_ref]. Thus OhrR binding sites are located within the 58 bp HpaII fragment. None of the DNA fragments generated by BssHII (54 and 59 bp) or MseI (47, 50 and 16 bp, the last not detected on the gel) were shifted in the presence of OhrR [fig_ref] Figure 3: Localisation of OhrR binding sites [/fig_ref]. The unique BssHII and both MseI sites are located within the 58 bp HpaII region, which suggests that OhrR binding site is located within the 16 bp MseI fragment or overlaps its extremities and overlaps the BssHII site. Two imperfect palindroms [fig_ref] Figure 3: Localisation of OhrR binding sites [/fig_ref] are located within the 58 bp HpaII region. Moreover MseI and BssHII sites overlap these motifs. Motif 1 (GCAAAT-TAATTTTG) and motif 2 (GCAAATTGCTTTGC) look like the OhrR binding site GCAATT-AATTCG found in other bacteria [bib_ref] Mongkolsuk S: ohrR and ohr are the primary sensor/regulator and protective genes..., Chuchue [/bib_ref] [bib_ref] Dual role of OhrR as a repressor and an activator in response..., Oh [/bib_ref] [bib_ref] OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant..., Fuangthong [/bib_ref] [bib_ref] Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves..., Sukchawalit [/bib_ref]. Motif 1 and motif 2 are adjacent as observed for OhrR binding sites of B. subtilis [bib_ref] OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant..., Fuangthong [/bib_ref] , A. tumefaciens [bib_ref] Mongkolsuk S: ohrR and ohr are the primary sensor/regulator and protective genes..., Chuchue [/bib_ref] , S. coelicolor [bib_ref] Dual role of OhrR as a repressor and an activator in response..., Oh [/bib_ref] and X. campestris [bib_ref] Complex regulation of the organic hydroperoxide resistance gene (ohr) from Xanthomonas involves..., Sukchawalit [/bib_ref]. To further analyse OhrR binding, 60 bp DNA fragments containing in their centre 17 nt corresponding either to motif 1 or motif 2 were synthesised. The OhrR protein was found to bind to both fragments. Mutations were introduced in motif 1 to confirm the importance of this sequence. The modification of GC to AA or AAA to CCC in one half of the palindrome abolishes the binding of OhrR to the DNA fragments (Figure3C). ## Modulation of ohrr activity by oxidation S. meliloti OhrR protein contains two cysteine residues conserved at the same position than in OhrR of X. Campestris, allowing the possibility to form inter-subunit disulfide bonds upon oxidation. Purified OhrR was treated with CuOOH, H 2 O 2 or DTT and the products were analysed by non reducing SDS-PAGE . In the presence of DTT, S. meliloti OhrR protein migrated as a band of an apparent MW of 15 kDa (the calculated molecular mass being 17.5 kDa). Incubation of the protein with CuOOH or H 2 O 2 led to the formation of a band of 30 kDa corresponding to the dimeric form. The formation of the dimer was reversed by an excess of DTT. Thus, as observed in X. campestris [bib_ref] Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor..., Panmanee [/bib_ref] , the oxidation of OhrR induces a reversible bonding between the two subunits of the protein . Binding of OhrR to ohr-ohrR intergenic region was suppressed when 10 mM H 2 O 2 was added to the binding mixture. Binding was recovered after addition of an excess of DTT. Thus only the reduced form of OhrR was able to bind DNA . ## Ohr strain forms fix + nodules in alfalfa The sensitivity of S. meliloti ohr mutants to OHPs is potentially relevant to symbiosis since legume root cells respond to rhizobial infection with an enhanced production of ROS [bib_ref] Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction, Santos [/bib_ref] [bib_ref] Nod factor induction of reactive oxygen species production is correlated with expression..., Ramu [/bib_ref]. To test the effect of ohr mutation on nodulation and nitrogen fixation, one week old seedlings of Medicago sativa were inoculated with either the S. meliloti ohr mutant or the parental strain. Plants were grown in nitrogen-deprived medium. Five weeks after the inoculation, plants were visually screened for nodulation by observing the root system. A highly efficient nodulation was observed on plants inoculated with either ohr or parental strains. No significant difference between dry weights of plant shoots was observed. The inoculated plants had green leaves and comparable number of nodules, whereas the non-inoculated control plants were smaller, with yellow leaves and significantly lower dry weight. Nodules from plants inoculated with the ohr mutant were crushed and the bacteria recovered by plating on MSY plates before assayed for gentamycine resistance and OHP sensitivity. All the randomly selected colonies that were analysed were able to grow on gentamycine-containing plates and behaved like the original ohr mutant. Thus N 2 -fixing nodules formed on alfalfa were due to infection by the ohr mutant and not by revertants. In order to analyse ohr and ohrR expression in planta, β-galactosidase and β-glucuronidase activity were visualised by light microscopy on entire and sections of nodules from R7.16 (ohr-lacZ, ohrR-uidA, ohr + , ohrR + ) infected plants . No staining was observed in root hairs or infection threads. Nodule staining co localises with pink coloration of leghemoglobin, corresponding to nitrogen fixation zone (data not shown). Thus, in spite of the absence of a nodulation defect of ohr strain, both ohr and ohrR genes were expressed during nodulation. This result is in accordance with the detection of Ohr protein in nodules in proteomic studies [bib_ref] Sinorhizobium meliloti metabolism in the root nodule: a proteomic perspective, Djordjevic [/bib_ref]. # Discussion In this study, we analysed the role of ohr and ohrR genes in S. meliloti. As many bacteria, S. meliloti must survive oxidative stress generated by the environment or during symbiosis. ROS attack of cellular membranes generates a cascade of radicals leading to the formation of OHPs [bib_ref] Pathways of oxidative damage, Imlay [/bib_ref]. Moreover, OHPs are produced by plants as part of the defence response against bacteria [bib_ref] Active oxygen in plant pathogenesis, Baker [/bib_ref] [bib_ref] Lipid peroxidation in cotton, Xanthomonas interactions and the role of lipoxygenases during..., Jalloul [/bib_ref]. Organic peroxides are potent effectors of ohr system in bacteria [bib_ref] Novel roles of ohrR-ohr in Xanthomonas sensing, metabolism, and physiological adaptive response..., Klomsiri [/bib_ref]. Ohr is not essential for nodulation. Bacteria containing ohr mutations formed effective nodules, suggesting that S. meliloti does not undergo OHP stress during nodulation or that other enzymes detoxify OHP like AhpC (a putative ahpC gene: SMb20964 was annotated) as described in X. campestris [bib_ref] Evaluation of the roles that alkyl hydroperoxide reductase and Ohr play in..., Vattanaviboon [/bib_ref]. The redundancy of enzymatic activities was also described for catalases in S. meliloti; only strains affected at least for two catalases are compromised in symbiosis [bib_ref] Expression of the bacterial catalase genes during Sinorhizobium meliloti-Medicago sativa symbiosis and..., Jamet [/bib_ref]. Both ohr and ohrR are specifically induced by OHPs and are expressed in nodules but no OHP detection was reported, so we could not exclude the existence of other compounds inducing ohr and ohrR. Like in many bacteria, ohr is located at the immediate vicinity of its regulator: ohrR (SMc00098). This ORF These two proteins share 40% amino acid identity and are structurally similar [bib_ref] Structure of an OhrR-ohrA operator complex reveals the DNA binding mechanism of..., Hong [/bib_ref] [bib_ref] Structural mechanism of organic hydroperoxide induction of the transcription regulator OhrR, Newberry [/bib_ref]. Nevertheless, they differ in their peroxide sensing mechanisms. The B. subtilis OhrR protein family contains only one cysteine residue. Depending on the oxidant, OhrR gives reversible oxidised derivatives or functions as a sacrificial regulator [bib_ref] Oxidant-dependent switching between reversible and sacrificial oxidation pathways for Bacillus subtilis OhrR, Soonsanga [/bib_ref]. The X. campestris OhrR possesses another important cysteine (C127). The initially oxidized cysteine (C22) forms intersubunits disulfide bonds with the residue C127 on the second subunit of the dimer, leading to reversible inactivation of the protein [bib_ref] Novel organic hydroperoxide-sensing and responding mechanisms for OhrR, a major bacterial sensor..., Panmanee [/bib_ref]. The introduction of a second cysteine into B. subtilis OhrR (position 120 to 124) allows B. subtilis OhrR to function as X. campestris OhrR, protecting the protein against irreversible oxidation in presence of strong oxidants [bib_ref] Conversion of Bacillus subtilis OhrR from a 1-Cys to a 2-Cys peroxide..., Soonsanga [/bib_ref]. Comparison of S. meliloti OhrR protein with that of B. subtilis and X. campestris shows that S. meliloti protein keeps similar amino acid identity with both proteins (45 and 49% respectively). S. meliloti possesses two cysteines at the same position than OhrR of X. Campestris. We observed that protein oxidation led to the formation of a dimer and loss of DNA binding, these phenomena are reversed by DTT in vitro. Thus S. meliloti OhrR oxidation mechanism is similar to that described for OhrR of X. campestris. The expression of ohr and ohrR was assayed at the transcriptional level. Their expression was constant throughout growth and no induction during stationary growth phase was observed. Similarly, osmotic stress did not induce ohr or ohrR expression. These observations match with the expression of these genes in X. campestris, A. tumefasciens, B. subtilis, P. aeruginosa and S. coelicolor [bib_ref] Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with..., Mongkolsuk [/bib_ref] [bib_ref] Mongkolsuk S: ohrR and ohr are the primary sensor/regulator and protective genes..., Chuchue [/bib_ref] [bib_ref] Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to..., Mostertz [/bib_ref] [bib_ref] Dual role of OhrR as a repressor and an activator in response..., Oh [/bib_ref] [bib_ref] Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide, Palma [/bib_ref]. As previously observed in these bacteria, ohr and ohrR genes of S. meliloti were induced by tBOOH and CuOOH. H 2 O 2 was a poor inducer of ohr gene in S. meliloti. Induction of ohr by H 2 O 2 in other bacteria is contradictory. Western analysis and gene fusion assays showed that ohr is not induced by H 2 O 2 in A. tumefasciens, B. subtilis, P. aeruginosa and S. coelicolor [bib_ref] Mongkolsuk S: ohrR and ohr are the primary sensor/regulator and protective genes..., Chuchue [/bib_ref] [bib_ref] Genetic and physiological characterization of ohr, encoding a protein involved in organic..., Ochsner [/bib_ref] [bib_ref] Dual role of OhrR as a repressor and an activator in response..., Oh [/bib_ref] [bib_ref] OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant..., Fuangthong [/bib_ref] and only X. campestris ohr is slightly induced by H 2 O 2 [bib_ref] Identification and characterization of a new organic hydroperoxide resistance (ohr) gene with..., Mongkolsuk [/bib_ref]. Transcriptomic studies of H 2 O 2 stress response in B. subtilis [bib_ref] Transcriptome and proteome analysis of Bacillus subtilis gene expression in response to..., Mostertz [/bib_ref] and P. aeruginosa [bib_ref] Transcriptome analysis of the response of Pseudomonas aeruginosa to hydrogen peroxide, Palma [/bib_ref] showed in contrast an ohr induction. Induction of ohr requires the oxidation of OhrR. We observed that S. meliloti OhrR is oxidized by H 2 O 2 in vitro and did not bind to the operator when incubated with H 2 O 2 . Nevertheless, H 2 O 2 is a poor inducer of ohr in vivo and is not an inducer of ohrR expression. H 2 O 2 also causes a loss of B. subtilis OhrR binding to ohrA promoter in vitro while in vivo derepression of ohrA upon exposure to H 2 O 2 was not observed [bib_ref] The OhrR repressor senses organic hydroperoxides by reversible formation of a cysteine-sulfenic..., Fuangthong [/bib_ref] [bib_ref] OhrR is a repressor of ohrA, a key organic hydroperoxide resistance determinant..., Fuangthong [/bib_ref]. The role of H 2 O 2 in alfalfa during symbiosis is not restricted to plant defence against bacteria. It is also important for symbiotic process [bib_ref] Reactive oxygen species during plant-microorganism early interactions, Nanda [/bib_ref]. H 2 O 2 is necessary for cell wall formation and infection thread rigidity [bib_ref] Oxidative burst in alfalfa-Sinorhizobium meliloti symbiotic interaction, Santos [/bib_ref]. Production of H 2 O 2 was detected in root hairs, infection threads, infection and senescence zones but not in fixing zone [bib_ref] Localization of superoxide dismutases and hydrogen peroxide in legume root nodules, Rubio [/bib_ref]. The expression of ohr and ohrR was detected only in nitrogen fixing zone, thus they are not expressed constitutively and they are not induced by H 2 O 2 in planta. These data suggest that organic peroxides are produced in nodules so that Ohr protein plays a role during nitrogen fixation. # Conclusions Resistance to organic hydroperoxides has not been previously analysed in S. meliloti. We have demonstrated that Ohr protein is essential for S. meliloti to survive organic peroxide stress. The expression of ohr and ohrR genes in nodules suggests that the Ohr protein participates in organic peroxides detoxification within the nodule. # Methods ## Bacterial strains, plasmids, and culture conditions The bacterial strains used in this study are detailed in [fig_ref] Table 1: Bacterial strains and plasmids mutation was transduced into S [/fig_ref]. S. meliloti strains were grown aerobically at 30°C in the complex medium LBto an optical density at 570 nm (OD 570 ) of 1.5 to 1.8; they were then inoculated in minimal galactose aspartate salts medium (GAS) [bib_ref] Sucrose is a nonaccumulated osmoprotectant in Sinorhizobium meliloti, Gouffi [/bib_ref] or LB medium at an OD 570 nm of 0.1. E. coli strains were grown aerobically in LB medium at 37°C. For the selection of E. coli strains, ampicillin was added at 50 or 100 μg ml -1 , tetracycline at 10 μg/ml, chloramphenicol at 25 μg/ml, and neomycin or kanamycin at 50 μg/ml. For the selection of S. meliloti strains, streptomycin was used at 100 μg/ml, tetracycline at 5 μg/ml, and neomycin at 25 μg/ml. ## Dna manipulations and mutant constructions Standard protocols were used for DNA manipulations. b-glucuronidase and b-galactosidase assays β-glucuronidase and β-galactosidase assays were carried out as described [bib_ref] uidA antibiotic resistance cassettes for insertion mutagenesis, gene fusion and genetic constructions, Bardonnet [/bib_ref]. Specific activities are expressed as nanomoles of ortho-nitrophenol liberated per minute per milligram of protein. Protein concentration was determined by the method of Bradford with bovine serum albumin as a standard. Results are the mean of at least three independent experiments, and the standard deviation was less than 10%. ## Disk diffusion assay Cells were grown in LB medium to an OD 570nm of 0.4. 0.5 ml of cell suspension were mixed with 3 ml of soft agar (0.4%) and poured onto LB agar plates (20 ml). 10 μl of 0.1 M cumene hydroperoxyde (CuOOH), 0.5 M tbutyl hydroperoxide (tBOOH), 10 M H 2 O 2 or 50 mM menadione were loaded on 8 mm paper disks placed on top agar. Plates were incubated for 24 h at 30°C and the clear zone was measured. CuOOH, tBOOH and menadione solutions were made in 95% ethanol. 10 μl of ethanol produced no growth inhibition in this assay. ## Construction of a δohrr strain A 2,152 bp DNA fragment corresponding to the upstream region of ohrR was amplified on S. meliloti chromosomal DNA using the primers (GATCGGCCTC-GACCCATACG) and (CCTCGTCTAGATGTCAT TGTCG; introduces an XbaI restriction site in place of ohrR ATG initiation codon) and cloned in pGEMTeasy vector (Promega, La Jolla, CA) giving pD3083. A 1,468 bp DNA fragment corresponding to the downstream region of ohrR was amplified using the primers (AGCTCTAGAGCACCTGCAG; introduces an XbaI restriction site in place of ohrR stop codon) and (CAGCGCGTGTGGCGGCG). This amplicon was digested with XbaI and NsiI (genuine site) and cloned into pK18mobsacB vector [bib_ref] Small mobilizable multi-purpose cloning vectors derived from the Escherichia coli plasmids pK18..., Schafer [/bib_ref] between the XbaI and PstI sites, giving pD3001. pD3083 and pD3001 were linearised with XbaI and ligated in order to assemble ohrR-upstream and -downstream sequences. Then pGEMTeasy vector was deleted through an EcoRI digest, giving pD4116, and the GmR cassette of p34SGm [bib_ref] Plasposons: modular self-cloning minitransposon derivatives for rapid genetic analysis of gram-negative bacterial..., Dennis [/bib_ref] was inserted into the XbaI site, giving pD4244. This final construction carries the ohr-ohrR region where the ohrR open reading frame is replaced by a GmR cassette; it was introduced into S. meliloti Rm1021 strain by triparental mating and recombinants were selected for on MSY medium containing gentamycin and sucrose. Double crossing over recombinants were identified as neomycin sensitive strains and confirmed by PCR. The ## Inactivation of ohr A 4 kb chromosomal DNA fragment containing ohr and ohrR genes was amplified by PCR using the primers (GATCGGCCTCGACCCATACG) and (CAGCGCGTGTGGCGGCG) and cloned into pGEM-Teasy vector. The insert was recovered with EcoRI and transferred to the same site on pK18mobsacB vector. The ohr open reading frame was then inactivated by introducing into the unique NotI site the GmR cassette from pBBR1-MCS5 digested with NotI. The resulting plasmid pD8657 was introduced into Rm1021 strain and double crossing events were selected as before and confirmed by PCR. The mutation was transduced into Rm1021 strain using ΦM12, yielding R8.39. Construction of an ohr::Gm R , ΔohrR strain pD4116 carries the entire ohr sequence and a deletion of ohrR. The ohr gene was disrupted by introducing in its unique NotI site the GmR resistance cassette from pBBR1-MCS5 recovered through a NotI digest. The resulting pD5333was conjugated into Rm1021 strain and double crossing overs were selected as previously described and confirmed by PCR. Transduction of the mutations into Rm1021strain yielded R7.15. Construction of ohr::lacZ, ohrR::uidA into a wild type genetic background The 4 kb chromosomal fragment amplified for ohr inactivation contains two SalI sites near the 3'end of ohr and ohrR genes respectively. It was cleaved with SalI and the 980 bp DNA fragment containing the 5' regions of ohr and ohrR was introduced into the XhoI site of pTH1705 vector (not replicative in S. meliloti) [bib_ref] An integrated approach to functional genomics: construction of a novel reporter gene..., Cowie [/bib_ref]. In the resulting pD5455 plasmid two transcriptional fusions are generated: ohr::lacZ and ohrR::uidA. pD5455 was introduced into Rm1021 strain by triparental mating. Single crossing over events were selected as GmR strains. Recombination was confirmed by PCR. The transduction of the mutation into Rm1021 strain yielded R7.16. ## Analysis of ohr regulation by ohrr ohr::lacZ region was released from pD5455 using XbaI and SphI and introduced between the corresponding sites of pBBR1-MCS2 vector (replicative in S. meliloti), yielding pE1541. This plasmid was introduced by triparental mating into the wild type strain and ohrR mutant (R6.48) and β-galactosidase activities were assayed in both strains. ## Complementation plasmids The open reading frames of ohr and ohrR were amplified using the primers (GATCGGCCTCGACCC ATACG) and (CCTCGTCTAGATGTCATTGTCG) for ohr and (CGTCGATAAAGAAGCCTGTG) and (CAG CGCGTGTGGCGGCG) for ohrR. The amplicons were cloned into pGEMTeasy, released by EcoRI cleavage and introduced into the same site in pBBR1-MCS2 vector. The correct orientation allowing the expression of these genes under the control of lac promoter was selected. The corresponding plasmids pBBohr and pBBohrR were introduced into Rm1021 strain and the various mutants by triparental mating. ## Purification of ohrr protein The ohrR open reading frame was amplified by PCR using the primers (CGACAATGACATATGACGAGG) and (AGCTCTCGAGTCGACTACCG) and cloned in pGEMT. The insert was released as an NdeI-XhoI DNA fragment and introduced into the expression vector pET22b+ (Novagen) giving pETohrR where the ohrR ORF is fused to a 6his-tag at its 3' extremity. BL21(DE3) cells harbouring pETohrR were cultured in LB medium at 37°C until OD 570 nm of 0.8; isopropyl-β-D-galactopyranoside was then added to a final concentration of 1 mM. The culture was grown for an additional 4 h, and cells were harvested by centrifugation (5,000 × g, 10 min, 4°C). Bacterial cells were washed in TE (10 mM Tris pH 6.8, 1 mM EDTA) and resuspended in the same buffer with 1 mM phenylmethylsulfonyl fluoride. Cells were disrupted by three passages through a French press (1,200 PSI), and cell debris were removed by centrifugation at 4°C, 12,000 × g for 30 min. Proteins were loaded on a heparin column (GE heath care), followed by a wash (10 column volumes) with buffer A (25 mM Tris-HCl pH8, 25 mM NaCl, 2 mM EDTA, 1 mM DTT). Elution was performed with the same buffer containing 0.5 M NaCl. The eluted fractions were analysed by SDS-PAGE, and those containing OhrR were pooled and dialysed against buffer A. ## Gel mobility shift The intergenic region between ohr and ohrR was amplified by PCR using the primers (ATGATGT-CATTGTCGCAAATTC) and (CATGACAGTCTC CTTCCTTGTG) as a 113 bp DNA fragment. Complementary oligonucleotides were also used in gel mobility assay; they were annealed in 50 mM Tris-HCl pH8, 0.25 M NaCl, 1 mM EDTA. DNA probes (20 pmoles) were incubated with OhrR protein (0 to 100 pmoles) in 20 μl binding buffer (20 mM Tris-HCl (pH 8.0), 50 mM KCl, 1 mM EDTA, 50 μM bovine serum albumin) at room temperature for 10 min. Binding mixture was run on 6% polyacrylamide gel in Tris-borate buffer. Gels were stained with SYBR gold (Molecular Probes) in 10 mM TBE pH 8.0 buffer and revealed with a transilluminator at 312 nm. To oxidize OhrR, organic peroxides were added to the binding buffer; reduction of the protein was performed with DTT. ## Plant assays Medicago sativa L. var. Europe (alfalfa) was used as host plant for testing nodulation of S. meliloti strains according to [bib_ref] Exopolysaccharide-deficient mutants of Rhizobium meliloti that form ineffective nodules, Leigh [/bib_ref]. Surface-sterilized germinating seedlings were grown in test tubes on nitrogen-free medium. One week old plants were inoculated with 10 9 cells of wild type and ohr mutant of S. meliloti. Plants were analysed after 5 to 9 weeks of growth. ## B-galactosidase and b-glucuronidase detection in plants Nodules were fixed and stained as previously described [bib_ref] Rhizobium meliloti Genes Encoding Catabolism of Trigonelline Are Induced under Symbiotic Conditions, Boivin [/bib_ref] and observed by light microscopy. [fig] Figure 2: Induction of the expression of ohr and ohrR by peroxides. Cells were grown in LB medium to an OD 570 nm of 0.4. ohr::lacZ (β-galactosidase) and ohrR::uidA (β-glucuronidase) expression was analysed 2 and 3 h after OHP addition. No addition (closed diamonds), 0.25 mM CuOOH (closed triangles), 1.6 mM tBOOH (open squares), 10 mM H 2 O 2 (open circles). Enzymatic activities are expressed as nmole of substrate hydrolysed per min and per mg of protein. Results are the average of four independent experiments; the standard deviation is indicated by bars. [/fig] [fig] Figure 3: Localisation of OhrR binding sites. A-Restriction map of the 113 bp ohr-ohrR intergenic region used in gel mobility shift assay. The location of the initiator codon and translation direction of ohr and ohrR is indicated by a white arrow. The position of the two palindromic binding motifs Motif 1 (M1) and Motif 2 (M2) is indicated by black arrows. B-Gel mobility shift assay of the ohr-ohrR intergenic region and of its restriction fragments produced by BssHII, ApoI, HpaII or MseI. DNA (20 pmoles) was incubated in the presence (+) or in the absence (-) of 20 pmoles of OhrR. C-Binding of OhrR to Motif 1 and Motif 2 sequences. Gel shift assay of the intergenic region and the 60 bp double strand sequences containing at their centre the genuine 17 nt corresponding to Motif 1 and Motif 2, or mutated Motif 1 with AA in place of GC (Mut1 fragment) and CCC in place of AAA (Mut2 fragment). DNA (20 pmoles) was incubated with the indicated amount of OhrR in the presence of 1 mM DTT. [/fig] [fig] Figure 4, Figure 5: Oxidation promotes OhrR dimerisation and inactivation. (A) OhrR purified protein (20 nmoles) was incubated for 15 min with CuOOH (0.55 nM ) or H 2 O 2 (0.5 nM ) and then, when indicated, added with 0.5 mM DTT and incubated for another 15 min. (B) The DNA fragment (20 pmoles) corresponding to ohr-ohrR intergenic region was incubated with purified OhrR protein (20, 50 or 100 pmoles) in the presence of 0.5 nM H 2 O 2 and in the absence or in the presence of 0.5 mM DTT. Expression of ohr and ohrR in planta. Nodules were fixed and stained with 5-bromo-4-chloro-3-indolyl-beta-Dgalactopyranoside (β-galactosidase detection) (a,c) or 5-bromo-4chloro-3-indolyl-beta-D-glucuronate (β-glucuronidase detection) (b, d) and visualised by light microscopy. (a, b) whole nodules, (c, d) thin sections of stained nodules. The images are representative of 30 nodules analysed. [/fig] [table] Table 1: Bacterial strains and plasmids mutation was transduced into S. meliloti Rm1021 strain using ΦM12[53], yielding R6.48. [/table]

Calcium pyrophosphate deposition disease of the temporomandibular joint invading the middle cranial fossa: Two case reports # Introduction Tophaceous pseudogout, also known as calcium pyrophosphate deposition disease (CPPD), is a recurrent acute attack of arthritis caused by calcium pyrophosphate dihydrate crystals deposited in articular cartilage and surrounding tissues such as labra, synovia, articular capsules, and tendons. Its clinical manifestations can be similar to those of gout. CPPD most commonly occurs in large joints, such as the knee, hip, shoulder, and elbow, and rarely involves metacarpophalangeal joints, temporomandibular joints (TMJs), the skull base, or the spine . Pritzker et alfirst described pseudogout in the TMJ in 1976. The incidence of CPPD is positively correlated with age and often occurs in middle-aged and elderly people. The prevalence of CPPD is higher in women than in men, with a male-to-female ratio of 1:(2-7). CPPD is a benign crystalline joint disease with a variety of clinical manifestations. When the TMJ is involved, it can be manifested as facial swelling and pain, tinnitus, unilateral conductive hearing loss, limited mouth opening, etc.. Here, we introduce two cases of pseudogout of the TMJ destroying the skull base and infiltrating the middle cranial fossa. To our knowledge, such cases are rare, with a total of 9 cases having been reported in the literature. The purpose of our report was to focus on the invasiveness of CPPD to adjacent structures of the TMJ and discuss the relevant imaging features and differential diagnosis. ## Personal and family history The two patients did not have a personal or family history of related issues. ## Physical examination Case 1: Her right maxillofacial region was swollen, and a mass approximately 2 cm × 2 cm in size in the right preauricular region was hard upon palpation. She had slightly restricted mouth opening and a slight left deviation of the angle of the mouth; there was no tenderness in the parotid region on either side. ## Case 2: He had maxillofacial asymmetry. The left TMJ area showed a large mass approximately 4 cm × 4 cm in size, with obvious tenderness. The mass was hard, with poor mobility, and slightly adhered to surrounding tissues with unclear boundaries. The range of motion of the TMJ was acceptable bilaterally. ## Laboratory examinations Case 1: Her blood glucose concentration was 16.09 mmol/L, and the other blood test results were within normal limits. ## Case 2: The laboratory examination results (including electrolyte level, liver and kidney function, blood cell count, ferritin level, and parathyroid and thyroid function) were normal. ## Imaging examinations Case 1: Computed tomography (CT) showed an irregular calcified mass in the right TMJ with a CT value of 450-1120 HU and a clear boundary. The mass grew around the right condyle, and the bone of the mandibular head was destroyed. The mass destroyed the glenoid fossa and extended to the skull base. Magnetic resonance imaging (MRI) showed that the mass was T1WI hypointense and T2WI hypointense with inhomogeneous significant enhancement. It was further shown that the lesion was in contact with the dura mater on the inferior surface of the right temporal lobe. ## Final diagnosis The final diagnosis in both cases was tophaceous pseudogout of the TMJ. ## Treatment ## Case 1 The lesion was excised from the preauricular incision. The mass in the right TMJ eroded the right zygomatic arch and skull base bone. During the operation, the whole mass and part of the parotid gland were removed, and a small amount of cerebrospinal fluid leakage was found. Bone wax was used to fill the bone defect, a temporalis muscle flap was used to repair the articular disc, and temporomandibular arthroplasty was performed. ## Case 2 The lesion was resected using the preauricular approach. In the left TMJ, there was an irregular mass with an intact capsule and a clear boundary. The left zygomatic arch and skull base bone were destroyed. There was obvious adhesion between the mass and dura. The lesion, partial parotid gland and left zygomatic arch were resected, and the left TMJ was reconstructed. ## Outcome and follow-up ## Case 1 The patient's temporal pain was completely relieved, and the discomfort during mouth opening and chewing significantly improved. The problems of mouth angle deviation and tooth closure were greatly improved. One week after surgery, a CT scan showed that the mass in the right TMJ was absentand B). ## Case 2 The patient's left temporal pain and tinnitus were significantly alleviated, and no recurrence of the mass was observed one year later. # Discussion CPPD can occur in one or more joints . Tophaceous pseudogout may be related to Volume 9 Issue 11 metabolic disorders, heredity, aging and joint injury, especially metabolic disorders and genetic factors. The related diseases mainly include diabetes mellitus, hypertension, hypothyroidism, hyperparathyroidism, hyperuricemia/gout, hemochromatosis, nonspecific joint pain, and hypomagnesemia. These diseases lead to abnormal pyrophosphate metabolism. The proteoglycan of the articular cartilage is destroyed, and calcium and pyrophosphate are released into synovial fluid. Calcium combines with pyrophosphate to form calcium pyrophosphate molecular crystals, which are deposited in cartilage, ligaments, tendons and joints. It can cause joint surface abrasion and stimulate the joint to produce inflammatory reactions at the same time. Family genetic factors are mostly related to functional mutations in the ANKH gene. The ANKH gene encodes a membrane transporter that transports pyrophosphate produced by cartilage stromal cell metabolism from intracellular to extracellular regions via the cell membrane. When ANKH is mutated, transporter activity increases, which leads to increased extracellular pyrophosphate levels, further causing calcium pyrophosphate molecular crystals to deposit in the joint. In this report, one patient had a history of diabetes for more than 10 years, and the other patient did not have any special abnormalities. The imaging features were periarticular calcified masses involving articular cartilage. X-ray can show bone destruction and articular cartilage calcification of the TMJ. However, the sensitivity of this method is not high, which may lead to misdiagnosis and missed diagnosis . CT can clearly show the size of the lesion, calcification and extent of bone destruction. The mandibular condyle is most often involved, as well as the mastoid process and the greater wing of the sphenoid bone. Both cases in our study involved the temporal bone and the greater wing of the sphenoid bone, and one involved the middle ear mastoid process. MRI can better identify the extent of lesion involvement and the relationships between the lesion and articular disc, parotid gland, facial nerve and inferior temporal fossa. Articular cartilage calcification in CPPD often develops later than arthropathy on imaging, and MRI can detect lesions earlier than X-ray or CT . MRI showed a mass with abnormal signals in the articular cavity, which was mostly T1WI hypointense and T2WI hypointense with significant inhomogeneous enhancement. Masses can grow along articular cartilage, with cartilage and synovial sac involvement . CT is superior to MRI in demonstrating calcification and has suggestive value for disease diagnosis. This disease should be diagnosed on the basis of the common CT and MRI manifestations. Calcification of the articular disc has strong suggestive significance. Tophaceous pseudogout occurring in the TMJ has similar clinical or imaging findings to other diseases. It requires differential diagnosis from synovial chondromatosis (SC), neoplastic calcification, ossifying myositis, gout, chondroblastoma, and chondrosarcoma . Both cases were misdiagnosed as SC before the operation. SC is a nonneoplastic disease characterized by connective tissue metaplasia, resulting in synovial cartilage formation. SC of the TMJ is rare, but clinically, it resembles pseudogout . The key point of identification is that SC is characterized by the formation of small, multiple cartilaginous nodules in the joint space . In a previous study, one patient was misdiagnosed with a malignant tumor and underwent radiotherapy. Tumorous calcifications are mostly lobulated calcified masses in the soft tissues around the joints and generally do not involve the joint space. Calcification of chondrogenic tumors is usually heterogeneous, and the contour of the lesion is not clear on CT, which helps in the diagnosis. The serum uric acid level is high in gout patients, bone destruction is obvious, and the degree of calcification is mild. Gout usually occurs in the metatarsophalangeal joints of men. Ossifying myositis is associated with a history of trauma and is mostly located within muscles. The disease does not involve the joint space and rarely causes bone changes . These manifestations differ from the characteristics of the cases described in this paper. Although the imaging features of tophaceous pseudogout in the TMJ have certain specificity, other diseases cannot be completely excluded. The final diagnosis depends on a histopathological diagnosis. Lambrecht et al and related literature reports agree that the presence of diamond crystal morphology under a birefringence microscope is the gold standard for the diagnosis of CPPD. The treatment of pseudogout of the TMJ is selected according to the patient's symptoms and the severity of bone destruction. Conservative treatment, such as physical therapy, drug therapy (nonsteroidal anti-inflammatory drugs, colchicine, etc.) and joint lavage can be used for patients with mild symptoms and noninvasive lesions . Follow-up examinations are necessary. Surgical treatment is advocated when intolerable symptoms and/or erosive disease occur . Surgical resection has been shown to be effective in relieving symptoms and TMJ mobility. Condylectomy, discectomy, and total resection are often combined. Of the 9 reported cases of pseudogout involving the middle cranial fossa, 6 underwent surgical resection. The symptoms were relieved in all cases. In this study, both patients underwent surgical Volume 9 Issue 11 treatment, with good prognoses. Pseudogout is benign but invasive. Long-term follow-up after surgical resection indicate that the prognosis is good. Pseudogout occurring in the TMJ rarely invades the skull base or middle cranial fossa. Pseudogout should be considered when a calcified mass with or without bone destruction occurs in the TMJ with corresponding clinical symptoms. # Conclusion In conclusion, cases of TMJ pseudogout that invade the middle cranial fossa are rare, and the clinical manifestations of this disease are atypical. These features make it difficult to diagnose. Preoperative imaging is helpful in the diagnosis and treatment of the disease.

Epithelial-Myoepithelial Carcinoma of the Base of Tongue with Possible Lung Metastases Background. Epithelial-myoepithelial carcinomas are rare neoplasms usually arising from the salivary glands. There is limited evidence in the literature on their prognosis in the base of the tongue but other cases have resolved without recurrence. Methods. The patient underwent biopsies demonstrating the diagnosis of epithelial-myoepithelial carcinoma of the base of tongue and a PET scan showed multiple bilateral rounded pulmonary nodules. Results. The patient declined chemotherapy and radiotherapy to maximise his quality of life and passed away under management from palliative care several months later. Discussion. This is the only case in the literature of this type of carcinoma in the base of the tongue resulting in metastases and a poor prognosis. The case highlights the importance of checking for metastases in such lesions and their potentially serious outcomes if left untreated. # Introduction Epithelial-myoepithelial carcinoma is a rare neoplasm that usually arises from the salivary glands, comprising approximately 1% of all salivary gland tumours [bib_ref] Epithelial-Myoepithelial Carcinoma of the Salivary Glands, Vázquez [/bib_ref] [bib_ref] Epithelialmyoepithelial carcinoma of the tongue base: A case for the case-report and..., Peters [/bib_ref]. Since it was first described in 1972, there have only been 566 cases of epithelialmyoepithelial carcinoma described in the literature. Histologically, the tumour is biphasic with clear myoepithelial cells surrounding epithelial-lined ducts resembling intercalated ducts [bib_ref] Epithelialmyoepithelial carcinoma: A review of the clinicopathologic spectrum and immunophenotypic characteristics in..., Seethala [/bib_ref]. The largest series of cases to date was a recent multiinstitution review of 246 cases which showed a disease specific survival at 180 months of 80.7% [bib_ref] Epithelial-Myoepithelial Carcinoma of the Salivary Glands, Vázquez [/bib_ref]. The case presented is an uncommon presentation of epithelial-myoepithelial carcinoma of the base of the tongue with only four other cases found in the literature. ## Case Our case is of a 52-year-old gentleman who presented with restricted tongue movement, dysarthria, and dysphagia that developed progressively over several months. He mentioned this at the Ear Nose and Throat clinic during follow-up of surgical drainage of a mucocele from the pterygopalatine fossa six months before. He also reported 15 kg of weight loss in the preceding four months. He is a nonsmoker with no medical history except for osteomyelitis following a meniscal knee repair four years before. An MRI showed a 37 × 34 × 42 mm mass in the base of tongue extending to the geniohyoid muscle as well as the genioglossus muscle that was most concerning for squamous cell carcinoma as shown in [fig_ref] Figure 1: MRI of lesion in base of tongue [/fig_ref]. His PET/CT scan showed a large intensely FDG avid tongue mass arising in the posterior of the tongue within the body of the tongue and extrinsic muscles and extending posterosuperiorly into the region of the lingual tonsils and midline tongue superiorly. There was moderate FDG uptake in adjacent nodes consistent with metastases. Multiple bilateral rounded pulmonary nodules were present within the lungs bilaterally, the largest of which measured 5 mm. These showed no significant FDG uptake and were below the resolution of the PET/CT to accurately characterise. The lung lesions were too small for biopsy to confirm metastasis; however, it was judged to be metastatic given the rapidly progressive disease and bilateral neck metastases. The tumour was staged as T4N2cM1. Two tongue biopsies performed were inconclusive; however, the third biopsy a month later showed an infiltrating 2 Case Reports in Otolaryngology biphasic tumour with solid nests composed of epithelial and myoepithelial cells as shown in [fig_ref] Figure 2: Haematoxylin and eosin [/fig_ref]. The tumour cells also demonstrated perineural invasion as shown in [fig_ref] Figure 3: H&E stain demonstrating perineural invasion of the tumour [/fig_ref]. There was nuclear atypia and prominent mitoses present. Tumour cells were positive for SMA, p40, p63, CK5/6, EMA (patchy and luminal), CEA (luminal), and CD43. Ki-67 reveals a high proliferation index (60%). The diagnosis was epithelialmyoepithelial carcinoma with myoepithelial anaplasia. Subsequently, the patient was offered radical local treatment with radiotherapy and chemotherapy. The patient declined these so as to maximise his quality of life. He was referred to the palliative care service for continued care and over a year later required a percutaneous endoscopic gastrostomy (PEG) tube for feeding. Approximately 18 months after the diagnosis, the patient passed away from obstruction of the airway. # Discussion Epithelial-myoepithelial carcinoma is a rare low-grade neoplasm that was first characterised in 1972 [bib_ref] Diagnosis and ultrastructure of the tubular carcinoma of the salivary gland ducts...., Donath [/bib_ref]. They most commonly arise in the salivary glands with approximately 60-80% arising in the parotid glands [bib_ref] Epithelial-Myoepithelial Carcinoma of the Salivary Glands, Vázquez [/bib_ref] [bib_ref] Epithelialmyoepithelial carcinoma: A review of the clinicopathologic spectrum and immunophenotypic characteristics in..., Seethala [/bib_ref]. The mean age is around 60 and affects females at a ratio of 1.5 : 1 with a mean tumour size of approximately 29 mm [bib_ref] Epithelialmyoepithelial carcinoma: A review of the clinicopathologic spectrum and immunophenotypic characteristics in..., Seethala [/bib_ref]. Epithelial-myoepithelial carcinomas are primarily treated surgically, as shown in the largest study with all but 9 of the 207 patients undergoing surgery [bib_ref] Epithelial-Myoepithelial Carcinoma of the Salivary Glands, Vázquez [/bib_ref]. 85 of these patients (41.1%) received radiotherapy in addition to surgery; however, no survival benefit was noted for those who received radiotherapy compared to those who did not. Epithelial-myoepithelial carcinomas do not commonly have distant metastases and this case presents the possibility of lung metastasis, although unfortunately histological confirmation was not obtained. In a review of 58 patients with epithelial-myoepithelial carcinomas, only 3 patients (5.2%) had evidence of metastatic disease with only one case being a distant metastasis to the iliac bone [bib_ref] Epithelialmyoepithelial carcinoma: A review of the clinicopathologic spectrum and immunophenotypic characteristics in..., Seethala [/bib_ref]. In the largest review of 246 cases of epithelial-myoepithelial carcinoma, 11 patients (4.47%) had distant metastases; however, the locations were unspecified. There is only one case in the literature of pulmonary metastases which were discovered 14 years after a parotidectomy for epithelial-myoepithelial carcinoma of the salivary gland [bib_ref] Epithelial-myoepithelial carcinoma of salivary gland with metastasis to lung: Report of a..., Noel [/bib_ref]. The pathology in our case also showed a high proliferation index of 60% with Ki-67 staining. In contrast, a review of 61 epithelial-myoepithelial carcinomas found a mean proliferative index of 16.9% with a range from 0 to 50% [bib_ref] Epithelialmyoepithelial carcinoma: A review of the clinicopathologic spectrum and immunophenotypic characteristics in..., Seethala [/bib_ref]. There have been cases of epithelial-myoepithelial carcinoma dedifferentiating into a high-grade carcinoma (HGC) with a proliferation index of 67.1% in the HGC and 11.5% in the epithelial-myoepithelial portion [bib_ref] Dedifferentiated epithelial-myoepithelial carcinoma of the parotid gland: a rare case report of..., Kusafuka [/bib_ref]. It is also rare for epithelial-myoepithelial carcinoma to develop in the base of tongue with only 4 other cases in the literature to our knowledge [bib_ref] Epithelialmyoepithelial carcinoma of the tongue base: A case for the case-report and..., Peters [/bib_ref] [bib_ref] Epithelialmyoepithelial carcinoma of the base of tongue: pathology and management, Puri [/bib_ref] [bib_ref] Epithelial-myoepithelial carcinoma in the base of the tongue: a case report, Kumai [/bib_ref] [bib_ref] Epithelial-myoepithelial carcinoma in the ventral surface of the tongue, De Matos [/bib_ref]. Two of these patients received surgery and two received chemoradiotherapy. Therefore, this is, to our knowledge, the first case of epithelialmyoepithelial carcinoma in the base of tongue that has not been aggressively treated and instead given palliative support. A comparison between the known cases in the literature of epithelial-myoepithelial carcinoma in the tongue is summarised in [fig_ref] Table 1: Cases found in the literature of epithelial-myoepithelial carcinoma in the tongue [/fig_ref] and is a continuation of the review done by Peters et al. [bib_ref] Epithelialmyoepithelial carcinoma of the tongue base: A case for the case-report and..., Peters [/bib_ref]. Our case shows the potential malignancy of epithelialmyoepithelial carcinomas with distant metastasis to the lungs and lymph nodes. It is the second case in the literature of epithelial-myoepithelial carcinoma with metastases to the lungs and only the fifth case to occur in the tongue, to our knowledge. Although this is a rare presentation of the tumour, this case may help to guide patient decision-making and prognostic information in the future by demonstrating the potential severity of the disease. # Disclosure No funding was received for this paper. The case here has not been presented nor published elsewhere. The patient's information has been deidentified; however, unfortunately the patient has passed away before written consent could be obtained. [fig] Figure 1: MRI of lesion in base of tongue. [/fig] [fig] Figure 2: Haematoxylin and eosin (H&E) stain at ×40 magnification. [/fig] [fig] Figure 3: H&E stain demonstrating perineural invasion of the tumour. [/fig] [table] Table 1: Cases found in the literature of epithelial-myoepithelial carcinoma in the tongue. [/table]

The annual costs of cardiovascular diseases and mental disorders attributable to job strain in France Background: Work stress has become a major occupational risk factor in industrialized countries and an important economic issue. The objective was to estimate the annual costs of coronary heart diseases (CHD) and mental disorders (MD) attributable to job strain exposure according to Karasek's model in France for the year 2003 from a societal perspective.Methods:We produced attributable fraction estimates which were applied to the number of cases (morbidity and mortality) and the costs of CHD and MD. Relative risk estimates came from a systematic literature review of prospective studies. We conducted meta-analyses based on this selection of studies. Prevalence of exposure to job strain came from the national SUMER survey conducted in France in 2003. Costs included direct medical costs and indirect costs: production losses due to sick leaves and premature deaths.Results: Between 8.8 and 10.2% of CHD morbidity was attributable to job strain, and between 9.4 and 11.2% of CHD mortality was attributable to this exposure for men. Between 15.2 and 19.8% of MD was attributable to job strain for men, and between 14.3 and 27.1% for women. As a whole, between 450 000 and 590 000 cases of diseases and 910-1130 deaths were attributable to job strain for men. From 730 000 to 1 380 000 cases of diseases and from 150 to 280 deaths were attributable to job strain for women. The total number of sick leave days amounted from 5 to 6.6 million days for men, and from 8.5 to 16 million days for women. The total costs of CHD and MD attributable to job strain exposure ranged from 1.8 to 3 billion euros for the year 2003 (0.12-0.19% GDP). Medical costs accounted for 11% of the total costs, value of life costs accounted for 13-15% and sick leave costs for 74-77%. The cost of CHD was estimated at 113-133 million euros and the cost of MD was between 1.7 -2.8 billion euros in 2003.Conclusion: This study on the economic burden of diseases attributable to job strain in France provides relevant insights for policy-makers when defining public health priorities for prevention policies. # Background Work stress has become a major occupational risk factor in all industrialized countriesand an important economic issue. The high cost of work stress for employers, social security systems and society as a whole has been emphasized in numerous studies [bib_ref] Assessing the economic impact of stress-the modern day hidden epidemic, Kalia [/bib_ref] [bib_ref] The role of the workplace in the production and containment of health..., Shain [/bib_ref] [bib_ref] Costs and benefits of stress prevention in organisations, Liukkonen [/bib_ref] but detailed evaluations of these costs have been very seldom in the literature. A few rough estimates were produced without explaining the method used for such estimates. For example, costs of work stress were estimated at €20 trillion in Europe per year, but details on the estimation method were not available. Moreover, the discrepancy between estimates can be very large. This is partly due to the fact that work stress can be defined as a risk factor (related to working conditions and work organization) or as a health outcome related to mental disorders. For example, Hoel and alestimated that the costs of work stress and bullying may account for approximately 0.5-3.5% of GDP per year in 2000 in the UK (approx £4.9-34.1 billion), based on the hypothesis that these risk factors represented 30% of the overall cost of ill-health and accidents. The Health and Safety Executive in the UK estimated the cost of sickness absence due to stress, depression and anxiety perceived by the worker as being 'caused or made worse by work' (Labour Force Survey, UK) at £530 million in 2006. A way to evaluate the cost of work stress is to measure claims costs due to stress-related conditions at work. But this type of evaluation depends on national legislative frameworks which vary from one country to another. For example, in Australia, attempts have been made to reduce the costs of stress-related claims by modifying legislative thresholds on such claims [bib_ref] Work-related stress in Australia: The effects of legislative interventions and the cost..., Guthrie [/bib_ref]. In general, occupational exposures costs based on workers compensations only reflect a part of total costs, since occupational diseases or injuries have to be declared by workers [bib_ref] What percentage of workers with work-related illnesses receive workers' compensation benefits?, Biddle [/bib_ref] and covered by social welfare or insurance schemes to appear as claims [bib_ref] Cost of work-related injuries in insured workplaces in Lebanon, Fayad [/bib_ref]. Attributable fractions have been widely used in the literature to estimate the burden of diseases attributable to occupational exposures in terms of morbidity or mortality cases and disability-adjusted life years [bib_ref] Contribution of occupational risk factors to the global burden of disease -a..., Fingerhut [/bib_ref] [bib_ref] The global burden of selected occupational diseases and injury risks: Methodology and..., Nelson [/bib_ref] [bib_ref] Review of estimates of the global burden of injury and illness due..., Driscoll [/bib_ref] [bib_ref] Dying for work: The magnitude of US mortality from selected causes of..., Steenland [/bib_ref] [bib_ref] Epidemiologic estimate of the proportion of fatalities related to occupational factors in..., Nurminen [/bib_ref] [bib_ref] Quantitative estimates of work-related death, disease and injury in New Zealand, T&apos;mannetje [/bib_ref]. Attributable fraction can also be used to estimate the monetary cost of diseases attributable to occupational exposures but literature remains very sparse on this issue, except for some studies [bib_ref] Occupational injury and illness in the United States. Estimates of costs, morbidity,..., Leigh [/bib_ref] [bib_ref] Costs of occupational injuries and illnesses in California, Leigh [/bib_ref]. Studies are even less numerous when considering the costs of disease attributable to exposures to psychosocial risk factors at work [bib_ref] Modeling the economic burden of diseases imputable to stress at work, Bejean [/bib_ref]. However, precise and detailed economic evaluations of the costs associated to psychosocial work exposures are much needed to show the magnitude of the problem, and to provide guidance to policy makers when defining public health priorities and allocation of limited resources. A large number of epidemiologic studies have shown that exposure to work stress is a risk factor for various health outcomes, especially cardiovascular diseasesand mental disorders [bib_ref] Psychosocial work environment and mental healtha meta-analytic review, Stansfeld [/bib_ref] [bib_ref] Psychosocial factors at work and risk of depression: a systematic review of..., Bonde [/bib_ref] [bib_ref] The relation between work-related psychosocial factors and the development of depression, Netterstrom [/bib_ref]. Less conclusive results have been found for musculoskeletal diseases [bib_ref] Epidemiology of work related neck and upper limb problems: psychosocial and personal..., Bongers [/bib_ref] [bib_ref] Niedhammer I: Fractions of cardiovascular diseases, mental disorders, and musculoskeletal disorders attributable..., Sultan-Taieb [/bib_ref]. The leading work stress model in the literature is Karasek's job strain model [bib_ref] The Job Content Questionnaire (JCQ): an instrument for internationally comparative assessments of..., Karasek [/bib_ref]. This model is based on two dimensions: psychological demands (i.e. job demands, time pressure, conflicting demands, etc.), and decision latitude (i.e. both control over work, named decision authority, and possibility of developing skills, named skill discretion). According to this model, the most detrimental situation is defined by the combination of high levels of psychological demands and low levels of decision latitude, and is called job strain. Data on the impact of job strain on workers' health are available in the literature and exposure to job strain has been measured in a national survey, representative of the working population, in France in 2003. The aim of this article was to estimate the costs of cardiovascular diseases (CVD), and more precisely coronary heart diseases (CHD) and mental disorders (MD), i.e. two highly prevalent common mental disorders, depression and anxiety, attributable to job strain exposure in France in 2003 from a societal perspective, using a cost-of-illness approach [bib_ref] Cost-of-illness studies: a major headache, Drummond [/bib_ref]. A societal perspective is used to take account of all the costs associated with a disease, no matter who bears the costs, including health services and insurances, patients and the production system (lost production). This approach is considered as appropriate for decision making concerning public health policies [bib_ref] Perspectives in economic evaluation, Byford [/bib_ref] [bib_ref] Canadian Guidelines for Economic Evaluation of Pharmaceuticals, Torrance [/bib_ref]. # Methods ## Af data and calculation As defined by Nurminen and Karjalainen [bib_ref] Epidemiologic estimate of the proportion of fatalities related to occupational factors in..., Nurminen [/bib_ref] , attributable fractions (AF) are an estimate of the fraction of cases that is "attributable to an exposure in a population and that would not have been observed if the exposure had been non-existent". AF calculations are based on relative risk RR estimates (risks of disease or death due to exposure to the risk factor) and prevalence of exposure P e estimates (proportion of the population exposed to the risk factor) [bib_ref] The occurrence of lung cancer in man, Levin [/bib_ref] : [formula] AF ¼ P e RR−1 ð Þ= 1 þ P e RR−1 ð Þ ð Þ ð 1Þ [/formula] RR estimates were derived from a systematic literature review of prospective studies published in peer-reviewed journals between 1998 and 2008 (inclusive), i.e. a period centered around the year 2003. Inclusion criteria were a sufficient sample size (over 100 individuals) of a population in industrialized countries, with an explicit use of Karasek's model for exposure assessment and a precise measurement of health outcomes (clinically diagnosed or based on validated instruments). Only studies studying men and women separately and providing unadjusted (or age-adjusted) and multi-adjusted RRs/ORs were retained. This literature review excluded studies based on a job-exposure matrix known to underestimate RRs [bib_ref] Study of the validity of a job-exposure matrix for psychosocial work factors:..., Niedhammer [/bib_ref]. Fifteen prospective studies were selected, more details on the selection process of the literature review are available elsewhere [bib_ref] Niedhammer I: Fractions of cardiovascular diseases, mental disorders, and musculoskeletal disorders attributable..., Sultan-Taieb [/bib_ref]. In order to summarize the results of this literature review, we conducted meta-analyses based on this selection of studies. We retained 9 studies for CVD [bib_ref] Two alternative job stress models and the risk of coronary heart disease, Bosma [/bib_ref] [bib_ref] Perceived job stress and incidence of coronary events: 3-year follow-up of the..., Bacquer [/bib_ref] [bib_ref] Justice at work and reduced risk of coronary heart disease among employees:..., Kivimäki [/bib_ref] [bib_ref] Job strain and ischaemic disease: does the inclusion of older employees in..., Kivimäki [/bib_ref] [bib_ref] Job stress and major coronary events: results from the Job Stress, Absenteeism..., Kornitzer [/bib_ref] [bib_ref] A prospective study of job strain and coronary heart disease in US..., Lee [/bib_ref] [bib_ref] Psychological job demands increase the risk of ischaemic heart disease: a 14-year..., Netterstrom [/bib_ref] [bib_ref] Marital stress worsens prognosis in women with coronary heart disease: The Stockholm..., Orth-Gomer [/bib_ref] [bib_ref] Job strain and risk of cardiovascular events in treated hypertensive Japanese workers:..., Uchiyama [/bib_ref] among which 8 produced RRs for CHD and 1 study yielded RR estimates for CVD [bib_ref] Job strain and ischaemic disease: does the inclusion of older employees in..., Kivimäki [/bib_ref] , i.e., for a broader range of cardiovascular outcomes. We included 6 studies for MD [bib_ref] Gender differences in the effects from working conditions on mental health: a..., Bildt [/bib_ref] [bib_ref] Job strain and evolution of mental health among nurses, Bourbonnais [/bib_ref] [bib_ref] Job stress and depression symptoms in middle-aged workers-prospective results from the Belstress..., Clays [/bib_ref] [bib_ref] Psychosocial factors at work and subsequent depressive symptoms in the Gazel cohort, Niedhammer [/bib_ref] [bib_ref] Stress and depression in the employed population, Shields [/bib_ref]. Depression and anxiety were evaluated using either validated self-administered questionnaires, such as the Beck Depression Inventory (BDI), Center for Epidemiologic Studies Depression Scale (CES-D), General Health Questionnaire (GHQ), and Psychiatric Symptom Index (PSI), or standardized diagnostic interviews such as CIDI (Composite International Diagnostic Interview). We calculated two sets of summary RR estimates separately based (1) on unadjusted or age-adjusted RRs and (2) on multi-adjusted RRs. We produced summary estimates for men and women separately. We used a random effects method with inverse variance weighting, which is considered as more conservative than the fixed effects method [bib_ref] Psychosocial work environment and mental healtha meta-analytic review, Stansfeld [/bib_ref]. Two indicators of heterogeneity between studies were used: Cochran's Q statistic with a p value <0.05 for a significant heterogeneity and the I 2 statistic [bib_ref] Measuring inconsistency in meta-analyses, Higgins [/bib_ref]. We used Stata software for meta-analysis calculations. The data used for the estimate of prevalence of exposure P e to job strain came from the national SUMER survey which was conducted in France in 2003. This survey is a periodical cross-sectional survey conducted by the French Ministry of Labour (DARES) and including a selfadministered questionnaire, including Karasek's Job Content Questionnaire (JCQ). In total, 24,486 employees, 12,241 men and 10,245 women, selected on a random basis responded to the JCQ (response rate: 96.5%). The data of the SUMER survey were weighted to provide estimates that were representative of the French working population. As a result, the 2003 SUMER survey provides high quality data for exposure to job strain among the French national working population according to gender [bib_ref] Study of the validity of a job-exposure matrix for psychosocial work factors:..., Niedhammer [/bib_ref] [bib_ref] Propriétés psychométriques de la version française des échelles de la demande psychologique,..., Niedhammer [/bib_ref] [bib_ref] Importance of psychosocial work factors on general health outcomes in the national..., Niedhammer [/bib_ref] [bib_ref] The contribution of occupational factors to social inequalities in health: findings from..., Niedhammer [/bib_ref]. The estimates for the prevalence of job strain exposure obtained from the SUMER survey were 19.6% for men, 28.2% for women, and 23.2% for the total population. We produced a range of attributable fraction (AF) values. The AF low range value for each disease was calculated using multi-adjusted RR summary estimates in formula (1) and the AF high range value was based on age-adjusted RR summary estimates. In this calculation, we assumed that ORs were satisfactory estimates for RRs, which allowed to use ORs for AFs calculation in formula (1). We computed confidence intervals at 95%, based on 100 000 simulated distributions of P e and RR, with the hypothesis that RR and P e follow independent normal distributions. Confidence intervals were calculated with the mean and standard deviation from the simulated values of AFs. We used SAS software for simulation calculations. ## Data on prevalence of diseases We considered all national surveys available in France for the number of cases (morbidity and mortality) of CVD and MD. Inclusion criteria were a definition of disease corresponding to health outcomes used in our summary RR estimates, the availability of data for men and women separately, the availability of data for workingage population, and the survey had to be conducted in 2003 or as close as possible to 2003. ## Chd morbidity data Since most of the selected studies for CVD produced RRs for CHD (8 studies out of 9), we retained prevalence and costs data on CHD rather than CVD to ensure consistency between data used in our estimation. We used the prevalence of CHD (CIM 10, codes I20-I25) from the database of the Health and Social Benefits Survey (Enquête Santé et Protection Sociale) conducted in France in 2004 by IRDES (Institut de Recherche et Documentation en Économie de la Santé). Weighted prevalence data were computed and provided by IRDES for men (1.38%) and women (0.37%) from 18 to 64 years. We assumed a stable prevalence of CHD between 2003 and 2004 in France. ## Chd mortality data We used mortality data in 2003 from the database of the Epidemiological Center for the Medical Causes of Death (Centre d'Épidémiologie sur les Causes Médicales de Décès, CEPIDC). It provides the number of deaths from CHD (CIM 10, codes I20-I25) for men and women from 20 to 64 years. ## Md morbidity data We computed the prevalence of MD (depression and anxiety) from a French national survey, the Ten-yearly Health survey 2002-2003 (Enquête Décennale Santé). Data were weighted by age and gender. MD were defined by a score obtained to CES-D equal or higher than 16. The prevalence of MD for the population from 18 to 64 years was 15.5% (95% confidence interval: 14.6%-16.4%) for men and 27.4% (95% confidence interval: 26.4%-28.4%) for women in France. ## Md mortality data We used data about the prevalence of suicide in 2003 provided by the Epidemiological Center for the Medical Causes of Death (Centre d'Épidémiologie sur les Causes Médicales de Décès, CEPIDC) for men and women from 20 to 64 years. Based on an analysis of the literature [bib_ref] Mental disorders in young and middle aged men who commit suicide, Isometsa [/bib_ref] [bib_ref] Suicides: mieux comprendre pour prévenir avec plus d'efficacité, Michel [/bib_ref] , we assumed that from 54 to 64% of suicides were due to depression. A sensitivity analysis was conducted to take account of these two percentages. Another sensitivity analysis was conducted using a more restrictive definition of mental disorders based on depression alone. For this analysis, we used depression morbidity data produced by ESEMED study (European Study of the Epidemiology of Mental disorders) which was based on a standardized diagnostic interview (WMH-CIDI: World Mental Health-Composite International Diagnostic Interview). According to this survey, the prevalence of depression was 4.7% (95% confidence interval: 3.6%-5.8%) for men and 8.5% (95% confidence interval: 7.1%-9.9%) for women in France. We computed AF estimates for depression using job strain RR estimates provided by Shields's [bib_ref] Stress and depression in the employed population, Shields [/bib_ref] study. It was the only study in our literature review based on a measure of depression using a standardized diagnostic interview (CIDI) and producing both crude and multi-adjusted RR estimates. ## Cost data and calculation Costs C were estimated from a societal perspective according to a prevalence approach and based on French data. They included medical costs M of CHD and MD and indirect costs: production losses due to sick leaves S and to premature deaths D attributable to job strain exposure, according to the following formula: [formula] C ¼ X CVD;MD i M i þ S i þ D i ð Þ AF ið2Þ [/formula] where AF i is the attributable fraction corresponding to disease i (CHD or MD). Data on production losses due to early retirement and to presenteeism were not available. For the data on medical and indirect costs of diseases we conducted a systematic review of French and international databases, reports (EUROHEED-CODECS, DREES, IRDES, CNAMTS, Base BDSP, OECD, WHO) and articles published in peer reviewed journals through Medline interrogations. Indirect costs were calculated on the basis of the human capital hypothesis [bib_ref] Estimating the cost of illness, Rice [/bib_ref] , by multiplying the number of lost days because of illness with the Gross Domestic Product per capita and per working day (GDP cd ) for the year 2003 in France. The value of production losses due to premature death is estimated to take account of the lost production due to the number of years lost between the age of death and the average retirement age, as shown in formula (3): [formula] VLY ¼ GDP cd X a N a X R−a y¼1 1 þ g ð Þ y 1 þ r ð Þ y !ð3Þ [/formula] Where VLY is the value of lost years of production due to disease (CHD or MD), GDP cd is the French Gross Domestic Product per capita and per working day, N a is the number of deaths due to the disease at the age a in the French population, R-a is the number of years lost between the age of death a and the average retirement age R, g is the annual growth rate of GDP and r the discount rate. A discount rate of 5% and a growth rate of 2% were posited, as generally assumed. A sensitivity analysis was conducted to take account of the two range values for the number of suicides due to depression in the literature (54% or 64%) and the two values of attributable fractions which we computed (from multi or age-adjusted RRs). ## Chd medical costs According to Paris and al, CHD represented 16% of national consumption of medical care and goods for diseases of the circulatory system in France in 1998. And according to Fenina and al, diseases of the circulatory system represented 12.6% of the total consumption of medical care and goods in France in 2002. We assumed that average medical expenses for each case of CHD were the same between genders and age groups and a stable proportion of CHD medical expenses in total consumption of medical care and goods from 1998 to 2003 in France. ## Chd indirect costs Statistics from the public medical care system provided the number of sick leave days as prescribed by cardiologists in France in 2003. We made the assumption that the distribution of sick leave days between men and women was similar to the distribution of CHD cases between genders. ## Md medical costs According to Paris and al, depression represented 1.05% of the total consumption of medical care and goods in France in 1998. We assumed that medical expenses for each case of MD were the same between genders and age groups and a stable proportion of MD medical expenses in total consumption of medical care and goods from 1998 to 2003. ## Md indirect costs A study by Morvan et al. [bib_ref] Saint-Denis: Institut National de Prévention et d'Education pour la Santé (INPES), Morvan [/bib_ref] , on the basis of a national survey conducted in France in 2005 (Baromètre Santé 2005), produced the proportion of mild, average and severe episodes of depression in the population suffering from depression and the corresponding number of sick leave days per year. According to this survey, 17.8% of persons suffering from mild depressive episodes take an average of 10.4 sick leave days per year. 26.2% of persons suffering from average depressive episodes take an average of 49.2 sick leave days per year. And 48.9% of persons suffering from major depressive episodes take an average of 108.1 sick leave days per year. We assumed that the distribution of sick leave days between men and women was similar to the distribution of depression cases between genders. We also assumed that the number of sick leave days was stable between 2003 and 2005. Health reasons for sick leaves are not available in health insurance databases in France because the medical cause of absence is covered by confidentiality. # Results Meta-analysis results are shown in . Summary RR estimates for CHD morbidity were significant for .2% of CHD morbidity was attributable to job strain. Attributable fractions (AF) for CHD mortality were 9.4-11.2% for men. AFs for CHD morbidity and mortality for women yielded non significant values [fig_ref] Table 2: Fractions of coronary heart diseases [/fig_ref]. Therefore, we could not infer the burden of CHD attributable to job strain exposure for women, for lack of significant RR data. Between 15.2 and 19.8% of MD were attributable to job strain for men, and between 14.3 and 27.1% for women, these two AFs being significant. AF estimates and 95% confidence intervals are shown in [fig_ref] Table 2: Fractions of coronary heart diseases [/fig_ref]. Between 22 071 and 25 743 cases of CHD were attributable to job strain exposure, and between 442 and 524 deaths among men. For MD, between 430 470 and 559 215 cases for men and 727 266-1 378 718 cases for women were attributable to job strain exposure. Between 619 and 894 deaths (suicides associated with depression) for men and women were attributable to this exposure. The number of sick leave days lost because of CHD attributable to job strain went from 55 015 to 64 167 days. For MD, the number of lost working days amounted from 5 016 529 to 6 516 864 days for men and from 8 475 264 to 16 067 034 days for women. The total costs of CHD and MD attributable to job strain exposure ranged from approximately 1.8 to 3 billion euros per year in France. This cost included medical costs, the value of sick leave days lost because of disease, and the value of life years lost because of premature death. The cost of CHD was estimated at 113-133 million euros. The cost of MD was around 1.7 and 2.8 billion euros. Results are shown in [fig_ref] Table 4: Costs of diseases imputable to job strain en France in 2003 [/fig_ref]. We computed a sensitivity analysis based on a less conservative hypothesis for the percentage of suicides associated with depression, according to data available in the literature [bib_ref] Mental disorders in young and middle aged men who commit suicide, Isometsa [/bib_ref] [bib_ref] Suicides: mieux comprendre pour prévenir avec plus d'efficacité, Michel [/bib_ref]. Assuming that 64% of suicides were associated with depression instead of 54%, the number of deaths related to MD attributable to job strain was between 556 and 723 for men, and between 178 and 337 for women. As a result, the total number of deaths increased by 18.5% and the total costs increased by around 2%. Therefore, this alternative hypothesis had a limited impact on the total costs attributable to job strain exposure. We also conducted a more conservative analysis based on a restrictive definition of mental disorders, including depression only. RR estimates for depression were derived from Shields's study [bib_ref] Stress and depression in the employed population, Shields [/bib_ref] and were from 2.9 to 3.3 for men and from 1.2 to 2 for women, with a nonsignificant multi-adjusted RR estimate for women. Thus, available data did not allow the calculation of AF based on multi-adjusted RR estimate for women. For men, 27.2-31.1% of depression was attributable to job strain and 22% for women. Between 232 308 and 266 007 cases of depression for men and 346 172 cases of depression for women were attributable to job strain exposure. The number of lost working days because of depression attributable to job strain amounted from 2 707 220 to 7 134 095 days for men and women. The cost of depression attributable to job strain was between 780 million and 1.6 billion euros. # Discussion Between 8.8 and 10.2% of CHD morbidity was attributable to job strain, and between 9.4 and 11.2% of CHD mortality was attributable to this exposure for men. Between 15.2 and 19.8% of MD (depression and anxiety) was attributable to job strain for men, between 14.3 and 27.1% for women. As a whole, between 450 000 and 590 000 cases of diseases and 910-1130 deaths were attributable to job strain for men. From 730 000 to 1 380 000 cases of diseases and from 150 to 280 deaths were attributable to job strain for women. The number of deaths attributable to job strain is approximately twice the Summary relative risks (RR) and odds-ratios (OR) on the association between job strain and coronary heart diseases (CHD) and mental disorders (MD) The cost of CHD was estimated at 113-133 million euros and the cost of MD was between 1.7 and 2.8 billion euros. Thus MD accounted for approximately 94-96% of the total costs attributable to job strain exposure. This is due to a higher prevalence of the disease among the population of working age, higher levels of attributable fractions, and also a high amount of sick leave days due to MD. The sensitivity analysis conducted on a more restrictive definition of mental disorders including depression only, produced much lower values in terms of number of cases and costs. This calculation can be considered as very conservative. Indeed, the definition of mental disorders was restricted to clinical depression, and thus excluded anxiety but also symptomatic cases of depression and anxiety. Furthermore, the RR estimates were based on only one available prospective study that produced RR estimates using a standardized diagnostic interview (CIDI) [bib_ref] Stress and depression in the employed population, Shields [/bib_ref]. This study yielded non-significant multi-adjusted RR estimates for women, which did not allow to calculate a low range value of AF estimates. Some limitations of the study must be pointed out. This estimate of the cost of diseases attributable to job strain exposure may be more under-estimated than over-estimated. We did not produce AF estimates of CHD morbidity and mortality for women since available data were very scarce and the rare included studies yielded non-significant RRs. Therefore the estimation of the cost of CHD attributable to job strain was provided for men only. Another way of interpreting the results may be that our estimates of costs may be null for women, something that may be a bit premature to conclude given the scarcity of the literature. Indeed, recent studies show an increase in CVD prevalence among women in the French population, which underlines the need for more investigations on the etiological role of job strain on CVD among women. The estimates did not take account of potential delayed effects of job strain exposure on cardiovascular or mental health. We included only CHD and MD in our study. The number of studies selected in our literature review was very small for musculoskeletal disorders (MSD) and concerned different locations (back, low back, neck, shoulder, upper extremity, elbow, hand, and wrist) [bib_ref] Niedhammer I: Fractions of cardiovascular diseases, mental disorders, and musculoskeletal disorders attributable..., Sultan-Taieb [/bib_ref]. Therefore we could not compute a summary OR and AF estimates for a specific MSD location which could be used for cost of illness estimates. Our study grasped only a part of the total amount of costs attributable to work stress since job strain is only one aspect of stress among other concepts, such as Effort-Reward Imbalance developed by Siegrist [bib_ref] The measurement of effort-reward imbalance at work: European comparisons, Siegrist [/bib_ref]. Cost data had also some limitations. Costs did not include production losses due to presenteeism and to early retirement for lack of data. Other cost categories could not be included in our estimates, such as the costs of informal care (provided by families and friends), intangible costs (cost of suffering, pain and discomfort) and the cost of job turnover for lack of data. We also limited the value of lost years of life to the value of lost production. We did not take account of the total losses of social welfare due to deaths through an estimation of willingness to pay or value of a statistical life for instance, for lack of available data. For all these reasons, our estimates can be considered as conservative. In addition, we made a number of assumptions in our calculations and these assumptions may not be verified easily. For example, we assumed that the RR of mental disorders associated with job strain was the same for morbidity and mortality, something that may be consistent for cardiovascular diseases but was not checked for mental disorders, the literature being rare on this topic [bib_ref] Job stress as a preventable upstream determinant of common mental disorders: A..., Lamontagne [/bib_ref]. Another example, the proportion of suicides due to depression was estimated to be 54%-64% of the total number of suicides in the general population but it was not possible to check whether these figures may be used for the working population. This study has also several strengths. Our metaanalyses for RR estimates showed a satisfactory homogeneity between studies, except for age-adjusted ORs of MD for women which yielded a significant level of heterogeneity. Overall, the risk of heterogeneity between studies was mitigated by the fact that we included only high quality studies in our systematic review of the literature, with similar exposure and with statistical analyses allowing the estimation of RRs. The data we used are highly consistent: RR, prevalence of exposure, prevalence of disease and cost data are based on the same definition of disease and exposure. To ensure such consistency, we conducted a systematic review of the literature for disease prevalence and costs data in France and we produced data for each gender separately. The only exception may be the use of mental disorders medical costs based on the medical cost of depression only that may underestimate the costs of mental disorders attributable to job strain. We performed a sensibility analysis including depression only to provide a very conservative estimate of mental disorders costs attributable to job strain. The medical costs used in our study included out-of pocket payments by patients along with medical expenses paid by insurances, which is well appropriate for estimations from a societal perspective. We produced two range values of AF estimates with confidence intervals, based on a meta-analysis of RRs. Our calculation method had several strengths: we took account of multi-adjusted and age-adjusted estimates in our calculations, which produced the AF range values. And we combined this approach to meta-analyses to get more precise estimates of AFs based on available data in the literature. This method allowed us to encompass various high-quality RR estimates in our summary RRs, and at the same time to take account of a certain level of uncertainty regarding RRs, since age-adjusted and multiadjusted estimates yielded different values. Our summary RRs for CVD are consistent with those from the meta-analysis by Kivimaki et al. [bib_ref] Epidemiologic estimate of the proportion of fatalities related to occupational factors in..., Nurminen [/bib_ref] , who found estimates of 16% for men for the fractions of cardiovascular deaths attributable to job strain in Finland. Our AF estimates for MD are in line with those of LaMontagne et al. [bib_ref] Job strainattributable depression in a sample of working Australians: assessing the contribution..., Lamontagne [/bib_ref] who reported fractions of 13.2% for men and 17.2% for women attributable to job strain in Australia. Their fractions resulted from the summary OR estimates from Stansfeld and Candy's meta-analysis [bib_ref] Psychosocial work environment and mental healtha meta-analytic review, Stansfeld [/bib_ref]. Our results are higher than the costs of diseases attributable to job strain exposure in a previous study by Béjean et Sultan-Taïeb [bib_ref] Modeling the economic burden of diseases imputable to stress at work, Bejean [/bib_ref]. For the year 2000 in France, total costs amounted for 1.2-1.6 billion euros but included also musculoskeletal disorders. Differences can be explained by the fact that the fractions of MD for women were underestimated (4.8%) since they were based on a limited selection of OR estimates in the literature. The cost of depression attributable to job strain in Australia was estimated by LaMontagne et al.at 730 million dollars AUD (approx.. 510 million euros) in 2007, given that 1.54 million persons suffer from depression in the Australian workforce. It is however difficult to compare these results with ours since categories of costs included in the estimations are different: job turnover and presenteeism costs are included in LaMontagne et al.'s study, while indirect costs related to premature death (suicide) are excluded. Our results provide an evaluation at one point of time, allowing projections of the cost of job strain according to the evolution of working environments and the trend of prevalence of exposure to job strain. It also allows comparisons with other countries. Our summary estimates of RRs for CHD and MD could be used for AF calculations in other countries where the prevalence of exposure to job strain has been measured in the working population. # Conclusions This study on the economic burden of diseases attributable to job strain in France provides relevant insights for policy-makers when defining public health priorities for prevention policies. Diseases attributable to job strain and related costs are avoidable since effective intervention strategies to prevent job stress have been identified in several literature reviews [bib_ref] The psychosocial and health effects of workplace reorganisation. 1. A systematic review..., Egan [/bib_ref] [bib_ref] The psychosocial and health effects of workplace reorganisation. 2. A systematic review..., Bambra [/bib_ref]. Our results highlight potential economic implications of the development of such prevention policies. [table] Table 2: Fractions of coronary heart diseases (CHD) and mental disorders (MD) attributable to job strain in France (%) [/table] [table] Table 4: Costs of diseases imputable to job strain en France in 2003 (euros) CHD: coronary heart diseases, MD: mental disorders.those summarized in the meta-analysis by Stansfeld and Candy[30] (summary OR: 1.82, 95% CI: 1.06-3.10). Our estimates of attributable fractions for CVD mortality are more conservative than those reported byNurminen & Karjalainen [/table]

18F-Fluorocholine uptake matching CT lesions in the lungs of a patient clinically cured from COVID-19 syndrome F-Fluorocholine uptake matching CT lesions in the lungs of a patient clinically cured from COVID-19 syndromeF-Fluorocholine uptake matching CT lesions in the lungs of a patient clinically cured from COVID-19 syndrome Received: 15 April 2020 / Accepted: 7 June 2020 # Springer-Verlag GmbH Germany, part of Springer Nature 2020IMAGE OF THE MONTH 18 A 76-year-old man was referred to 18 F-fluorocholine (FCH) PET/CT for biochemical recurrence of prostate cancer (BRPC) after prostatectomy in 2004 (initial TNM: pT2bN0). Prostate-specific antigen (PSA) serum levels had been slowly rising (from 0.1 ng/mL in 2015 to 0.31 ng/mL in 2020). This patient presented with fever and cough 1 month before, considered as highly consistent with a COVID-19 infection by his general practitioner [bib_ref] Imaging and clinical features of patients with 2019 novel coronavirus SARS-CoV-2, Xu [/bib_ref] and a positive antibody testing confirmed the infection (immunoglobulin G = 7.67, positive if > 1.4). At the time of FCH PET-CT, he has been asymptomatic for over 1 week, and still is 2 months later. One hour after intravenous injection of 210 MBq of FCH, no focus evocative of BRPC was found. However, bilateral pulmonary foci were discovered (SUVmax 3.9). On CT, they matched ground-glass opacities and multifocal patchy consolidative opacities involving approximately 30% of the lungs, predominating in the peripheral inferior and posterior regions: typical CT features of COVID-19 infection [bib_ref] Imaging and clinical features of patients with 2019 novel coronavirus SARS-CoV-2, Xu [/bib_ref] [bib_ref] Correlation of chest CT and RT-PCR testing in coronavirus disease 2019 (COVID-19)..., Ai [/bib_ref] [bib_ref] Chest CT findings in coronavirus disease-19 (COVID-19): relationship to duration of infection, Bernheim [/bib_ref]. Bilateral mediastinum lymph nodes also took up FCH (SUVmax 3.8). All these lesions were not visible on a previous FCH PET-CT performed in 2018. COVID-19-induced lung lesions may take up 18 Ffluorodeoxyglucose [bib_ref] 18F-FDG PET/CT findings of COVID-19: a series of four highly suspected cases, Qin [/bib_ref] [bib_ref] Incidental findings suggestive of covid-19 in asymptomatic patients undergoing nuclear medicine procedures..., Albano [/bib_ref] and 18 F-fluorocholine as in this case. FCH was already known to reveal inflammatory conditions [bib_ref] 18F-choline in experimental soft tissue infection assessed with autoradiography and high-resolution PET, Wyss [/bib_ref] [bib_ref] Comparison of 18F-fluoro-deoxy-glucose,18F-fluoro-methyl-choline, and 18F-DPA714 for positron-emission tomography imaging of leukocyte accumulation..., Sarda-Mantel [/bib_ref] [bib_ref] Cancer imaging with fluorine-18-labeled choline derivatives, Kwee [/bib_ref]. FCH uptake by mediastinum lymph nodes is frequent but this usual pattern differs from the present images. The significance of those metabolically active lesions in a patient who clinically recovered from a COVID-19 infection is unknown: healing with a risk of lung fibrosis or subacute evolution with a risk of recurrence which did not occur within 2 months and of contamination? Male sex is associated with prolonged SARS-CoV-2 RNA shedding; thus, other cases of COVID-19 imaging patterns are likely to be discovered on FCH PET/CT in the future. This article is part of the Topical Collection on Image of the month * Léa Turpin [email protected] [bib_ref] Imaging and clinical features of patients with 2019 novel coronavirus SARS-CoV-2, Xu [/bib_ref]

Cognitive decline due to excess synaptic Zn2+ signaling in the hippocampus Zinc is an essential component of physiological brain function. Vesicular zinc is released from glutamatergic (zincergic) neuron terminals and serves as a signal factor (Zn 2+ signal) in both the intracellular (cytosol) compartment and the extracellular compartment. Synaptic Zn 2+ signaling is dynamically linked to neurotransmission and is involved in processes of synaptic plasticity such as long-term potentiation and cognitive activity. On the other hand, the activity of the hypothalamic-pituitary-adrenal (HPA) axis, i.e., glucocorticoid secretion, which can potentiate glutamatergic neuron activity, is linked to cognitive function. HPA axis activity modifies synaptic Zn 2+ dynamics at zincergic synapses. An increase in HPA axis activity, which occurs after exposure to stress, may induce excess intracellular Zn 2+ signaling in the hippocampus, followed by hippocampus-dependent memory deficit. Excessive excitation of zincergic neurons in the hippocampus can contribute to cognitive decline under stressful and/or pathological conditions. This paper provides an overview of the "Hypothesis and Theory" of Zn 2+ -mediated modification of cognitive activity. # Introduction Over 300 proteins require zinc to carry out their functions in microorganisms, plants, and animals. Zinc powerfully influences cell division and differentiation [bib_ref] The biological basis of zinc physiology, Vallee [/bib_ref] [bib_ref] Possible roles of zinc nutriture in the fetal origins of disease, Maret [/bib_ref] [bib_ref] Zinc in human health: effect of zinc on immune cells, Prasad [/bib_ref]. Zinc is also essential for the growth and functioning of the brain. Zinc transport from the plasma to the brain's extracellular fluid and cerebrospinal fluid is strictly regulated by the brain-barrier system, i.e., the blood-brain and blood-CSF barrier. The brain barrier system maintains zinc homeostasis in the brain [bib_ref] Movement of zinc and its functional significance in the brain, Takeda [/bib_ref] [bib_ref] Zinc homeostasis and functions of zinc in the brain, Takeda [/bib_ref]. Zinc homeostasis is critical for brain function [bib_ref] Zinc dyshomeostasis: a key modulator of neuronal injury, Capasso [/bib_ref] and is spatiotemporally altered in the process of neurological diseases [bib_ref] Metals in Alzheimer's and Parkinson's diseases, Barnham [/bib_ref]. Zinc is relatively concentrated in the hippocampus and amygdala [bib_ref] Involvement of glucocorticoid-mediated Zn 2+ signaling in attenuation of hippocampal CA1 LTP..., Takeda [/bib_ref]. Both regions are enriched with histochemically reactive zinc, as revealed by Timm's sulfide-silver staining method [bib_ref] Neurobiology of zinc and zinc-containing neurons, Frederickson [/bib_ref] [bib_ref] Zinc-containing neurons in hippocampus and related CNS structures, Frederickson [/bib_ref]. Histochemically reactive zinc is found predominantly in the presynaptic vesicles and serves as a signal factor (Zn 2+ signal) in both the cytosolic and extracellular compartments. Zn 2+ is released with glutamate in a calcium-dependent and impulse-dependent manner from glutamatergic (zincergic) neuron terminals . Zn 2+ released from these terminals modulates the activity of several important receptors, including the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptor, N-methyl-D-aspartate (NMDA) receptors, and γ -amino butyric acid (GABA) receptors in the extracellular compartment [bib_ref] Modulation of inhibitory and excitatory amino acid receptor ion channels by zinc, Smart [/bib_ref] [bib_ref] Zinc and cortical plasticity, Nakashima [/bib_ref] , and is taken up into post-synaptic neurons to serve as an intracellular signal factor. Glutamatergic (zincergic) circuits play a key role in cognitive map building structures such as the hippocampus [bib_ref] Zinc-positive boutons in the cerebral cortex of lizards show glutamate immunoreactivity, Martinez-Guijarro [/bib_ref] [bib_ref] Early histological maturation in the hippocampus of the guinea pig, Nacher [/bib_ref]. It has been estimated that approximately 20% of total brain zinc is histochemically reactive, based on the finding that the removal of zinc transporter-3 (ZnT3) protein, which is responsible for the movement of zinc from the cytoplasm into synaptic vesicles [bib_ref] ZnT-3, a putative transporter of zinc into synaptic vesicles, Palmiter [/bib_ref] , results in a 20% reduction of the total amount of zinc in the brain [bib_ref] Elimination of zinc from synaptic vesicles in the intact mouse brain by..., Cole [/bib_ref]. It is well known that the hippocampus and amygdala are involved in cognitive and emotional behavior. Synaptic plasticity such as long-term potentiation (LTP) is believed to be a key cellular mechanism involved in learning and memory and has been widely studied in relation to glutamatergic synapses in the brain, especially in the hippocampus [bib_ref] Expression of NMDA receptor-dependent LTP in the hippocampus: bridging the divide, Bliss [/bib_ref]. When information is processed in memory, glutamatergic neurons form a neural circuit in the hippocampus and the amygdala. Furthermore, it has been reported that plastic changes in hippocampal synapses occur activity-dependently during the performance of associative learning tasks [bib_ref] Plastic modifications induced by object recognition memory processing, Clarke [/bib_ref]. On the other hand, the activity of the hypothalamic-pituitaryadrenal (HPA) axis, i.e., glucocorticoid secretion, is linked to cognitive and emotional functions and can potentiate glutamatergic neuron activity [bib_ref] Glucocorticoids act on glutamatergic pathways to affect memory processes, Sandi [/bib_ref]. There is some evidence that the modification of synaptic Zn 2+ signaling by HPA axis activity, which is enhanced by stress and aging, is linked to cognitive and emotional behavior, and that abnormal modification may induce cognitive decline [bib_ref] Insight into zinc signaling from dietary zinc deficiency, Takeda [/bib_ref]. It is well known that abnormal Zn 2+ influx into post-synaptic neurons, which is induced by abnormal glutamatergic (zincergic) neuron activity, induces neuronal death and is involved in neurological disorders such as stroke/ischemia and temporal lobe epilepsy [bib_ref] The neurobiology of zinc in health and disease, Frederickson [/bib_ref] [bib_ref] The neurophysiology and pathology of brain zinc, Sensi [/bib_ref] [bib_ref] Insight into glutamate excitotoxicity from synaptic zinc homeostasis, Takeda [/bib_ref] [bib_ref] Ca permeable AMPA channels in diseases of the nervous system, Weiss [/bib_ref]. Therefore, the homeostasis of synaptic Zn 2+ signaling is critical in both functional and pathological aspects (Takeda, FIGURE 1 | Involvement of synaptic Zn 2+ dynamics in cognitive activity. An increase in intracellular Zn 2+ concentration, [Zn 2+ ] i , which is induced by an influx of extracellular Zn 2+ at zincergic synapses in the hippocampus, is involved in cognitive activity. Presynaptic glucocorticoid signaling, a non-genomic action, and post-synaptic glucocorticoid signaling, a genomic action, modify the degree of increase in intracellular Zn 2+ . It is also possible that catecholamines modify the degree through the activity of the β-adrenergic system. The degree of increase in intracellular Zn 2+ is linked to cognitive activity and excess intracellular Zn 2+ signaling, which can be induced by stress, is involved in cognitive decline. The excess might affect intracellular Ca 2+ signaling, which plays a key role for synaptic plasticity. 2011b; [bib_ref] Synaptic Zn 2+ homeostasis and its significance, Takeda [/bib_ref]. On the basis of recent evidence that excessive excitation of zincergic neurons in the hippocampus can contribute to cognitive decline under stressful and/or pathological conditions , this paper provides an overview of the "Hypothesis and Theory" of Zn 2+ -mediated modification of cognitive activity. ## Synaptic zn 2+ homeostasis Total zinc concentration in the adult brain reaches around 200 μM [bib_ref] Brain trace element concentrations in aging, Markesbery [/bib_ref]. Extracellular zinc concentration in the adult brain is estimated to be less than 1 μM [bib_ref] Zn( 2+ ): a novel ionic mediator of neural injury in brain..., Weiss [/bib_ref]. If zinc concentration in the brain's extracellular fluid is equal to that in cerebrospinal fluid [bib_ref] Cerebrospinal fluid trace element content in dementia: Frontiers in Aging Neuroscience www.frontiersin.org..., Hershey [/bib_ref] , it is around 150 nMapproximately one thousandth of total brain zinc concentration. In zincergic synapses, Zn 2+ concentration in the synaptic cleft is estimated to be higher than that in the brain's (extrasynaptic) extracellular fluid, because under hippocampal-slice-experiment conditions the regions where zincergic synapses are found are intensely stained by ZnAF-2, a membrane-impermeable zinc indicator [bib_ref] Inhibition of presynaptic activity by zinc released from mossy fiber terminals during..., Minami [/bib_ref]. The synaptic cleft is surrounded with the processes of astrocytes, which contribute to maintaining a steady concentration of zinc and neurotransmitters in the cleft. Interestingly, Zn 2+ level in the brain's extracellular fluid, which is estimated to be approximately 20 nM [bib_ref] Concentrations of extracellular free zinc (pZn)e in the central nervous system during..., Frederickson [/bib_ref] , is higher than that in the plasma (<1 nM; [bib_ref] The concentrations of free Mg 2+ and free Zn 2+ in equine..., Magneson [/bib_ref]. In the brain's extracellular fluid, the high ratio of Zn 2+ concentration to total zinc concentration appears to be associated with the synaptic Zn 2+ dynamics of the brain. There is some evidence that extracellular Zn 2+ serves as a pool for the zinc in the synaptic vesicle and is involved in synaptic Zn 2+ homeostasis [bib_ref] Responsiveness to kainate in young rats after 2-week zinc deprivation, Takeda [/bib_ref] , although the chemical form of this vesicular zinc is unknown. Basal Zn 2+ concentration is extremely low in the intracellular (cytosol) compartment (<1 nM; [bib_ref] Measurement of intracellular free zinc in living cortical neurons: routes of entry, Sensi [/bib_ref] [bib_ref] Insights into Zn 2+ homeostasis in neurons from experimental and modeling studies, Colvin [/bib_ref]. ZnT proteins such as ZnT1, ZnT3, and ZnT10, and Zrt-Irt-like proteins (ZIP) such as ZIP4 and ZIP6 are involved in the control of Zn 2+ levels in the cytosolic compartment, especially under static (basal) conditions Frontiers in Aging Neuroscience www.frontiersin.org 2010). Some of these transporters transport cytosolic Zn 2+ into a variety of subcellular organelles, including mitochondria, lysosomes, endosomes, and the Golgi apparatus, probably to maintain static Zn 2+ levels in the cytosolic compartment [bib_ref] Modulation of mitochondrial function by endogenous Zn 2+ pools, Sensi [/bib_ref] [bib_ref] Zinc-specific autometallographic in vivo selenium methods: tracing of zinc-enriched (ZEN) terminals, ZEN..., Danscher [/bib_ref] [bib_ref] Zinquin identifies subcellular compartmentalization of zinc in cortical neurons. Relation to the..., Colvin [/bib_ref]. On the other hand, it is possible that Zn 2+ release from subcellular organelles, which might be induced by synaptic glutamate signaling, is involved in Zn 2+ signaling [bib_ref] Zinc release from thapsigargin/IP3-sensitive stores in cultured cortical neurons, Stork [/bib_ref]. Zn 2+ levels other than vesicular zinc serving as Zn 2+ are estimated to be less than 5% of the total amount of Zn 2+ in the hippocampus and cerebral cortex [bib_ref] Dependence of the histofluorescently reactive zinc pool on zinc transporter-3 in the..., Lee [/bib_ref]. ZnT1 is a major Zn 2+ transporter in the plasma membrane and may be involved in cytosolic Zn 2+ homeostasis in neurons by transporting Zn 2+ from the somata to the extracellular space [bib_ref] Distribution of the zinc transporter ZnT-1 in comparison with chelatable zinc in..., Sekler [/bib_ref]. It has been reported that ZnT1 prevents excessive accumulation of Zn 2+ in the cytosolic compartment, resulting in the protection of neurons from Zn 2+ toxicity in neurological diseases such as transient forebrain ischemia [bib_ref] The increase in zinc levels and upregulation of zinc transporters are mediated..., Aguilar-Alonso [/bib_ref]. Tissue plasminogen activator, a secreted serine protease, is excitotoxic and increases lysosomal sequestration of increased Zn 2+ in the cytosolic compartment through interaction with ZIP4, which may also contribute to the protection of neurons from Zn 2+ toxicity [bib_ref] Tissue plasminogen activator alters intracellular sequestration of zinc through interaction with the..., Emmetsberger [/bib_ref]. The spatiotemporal control of Zn 2+ signaling via ZIP and ZnT maintains a steady-state environment in both the extracellular and cytosolic compartments [bib_ref] Molecular and genetic features of zinc transporters in physiology and pathogenesis, Fukada [/bib_ref]. ## Functional and neurotoxic zn 2+ signaling Zn 2+ concentration is increased in the synaptic cleft during the excitation of zincergic synapses, followed by an increase in the cytosol (intracellular compartment; . Released Zn 2+ is quickly taken up into presynaptic and post-synaptic neurons and astrocytes. Calcium channels such as calcium-permeable AMPA/kainate receptors are involved in Zn 2+ influx during neuronal excitation [bib_ref] Zn( 2+ ): a novel ionic mediator of neural injury in brain..., Weiss [/bib_ref] [bib_ref] Zn 2+ currents are mediated by calcium-permeable AMPA/kainite channels in cultured murine..., Jia [/bib_ref] [bib_ref] Role of zinc influx via AMPA/kainate receptor activation in metabotropic glutamate receptor-mediated..., Takeda [/bib_ref]. The increase in the extracellular concentration of Zn 2+ is dependent on the frequency of depolarizing stimulation [bib_ref] Mossy fiber Zn 2+ spillover modulates heterosynaptic N-methyl-Daspartate receptor activity in hippocampal..., Ueno [/bib_ref]. Therefore, the increase in intracellular concentration of Zn 2+ serving as a signal factor is closely correlated to zincergic neuron excitation [bib_ref] Synaptic Zn 2+ homeostasis and its significance, Takeda [/bib_ref]. Glutamate accumulates in the extracellular compartment due to excessive excitation of glutamatergic (zincergic) neurons. Excessive activation of glutamate receptors caused by excess extracellular glutamate leads to a number of deleterious consequences, including impairment of calcium buffering, generation of free radicals, activation of mitochondrial permeability transition, and secondary excitotoxicity [bib_ref] Glutamate uptake, Danbolt [/bib_ref] [bib_ref] Molecular mechanisms of excitotoxicity and their relevance to pathogenesis of neurodegenerative diseases, Dong [/bib_ref]. Glutamate excitotoxicity, a final common pathway for neuronal death, is observed in numerous pathological processes such as stroke/ischemia, temporal lobe epilepsy, Alzheimer's disease, and amyotrophic lateral sclerosis. An excess of extracellular Zn 2+ , which is induced under glutamate excototoxicity, is harmful; excessive Zn 2+ influx into post-synaptic neurons is involved in neurodegeneration under pathological conditions. Calciumpermeable AMPA receptors may play a key role in this Zn 2+ influx [bib_ref] Expression of Ca( 2+ )-permeable AMPA receptor channels primes cell death in..., Liu [/bib_ref] [bib_ref] Blockade of calcium-permeable AMPA receptors protects hippocampal neurons against global ischemia-induced death, Noh [/bib_ref] [bib_ref] Ca permeable AMPA channels in diseases of the nervous system, Weiss [/bib_ref]. Zn 2+ also plays a neuroprotective role in glutamate-induced excitotoxicity by activating pre-synaptic ATP-sensitive potassium channels and by inhibiting GABA transporter 4 [bib_ref] Zinc inhibits glutamate release via activation of pre-synaptic KATP channels and reduces..., Bancila [/bib_ref] [bib_ref] Zinc inhibition of gamma-aminobutyric acid transporter 4 (GAT4) reveals a link between..., Cohen-Kfir [/bib_ref]. It is estimated that the neuroprotective action of Zn 2+ occurs under conditions in which zincergic neurons are not excessively excited. Zn 2+ released from zincergic neuron terminals may also serve as a negative feedback factor against glutamate release [bib_ref] Inhibition of presynaptic activity by zinc released from mossy fiber terminals during..., Minami [/bib_ref] [bib_ref] Negative modulation of presynaptic activity by zinc released from Schaffer collaterals, Takeda [/bib_ref]. Therefore, the degree of increase in extracellular Zn 2+ is critical in both functional and neurotoxic aspects. ## Zn 2+ signaling and cognition Synaptic Zn 2+ signaling is involved in processes of synaptic plasticity such as LTP in the hippocampus and amygdala. Enhanced plasticity in zincergic synapses is associated with cortical modification after exposure to an enriched environment [bib_ref] Enhanced plasticity in zincergic, cortical circuits after exposure to enriched environments, Nakashima [/bib_ref]. The enhanced plasticity of zincergic synapses in the hippocampus underlies the acquisition of new motor and cognitive abilities (Delgado-García and [bib_ref] Differential contribution of hippocampal circuits to appetitive and consummatory behaviors during operant..., Jurado-Parras [/bib_ref]. These findings suggest that synaptic Zn 2+ signaling is involved in cognitive and emotional behavior through the modulation of synaptic plasticity such as LTP . Targeted deletion of the ZnT3 prevents vesicular Zn 2+ uptake [bib_ref] Elimination of zinc from synaptic vesicles in the intact mouse brain by..., Cole [/bib_ref] and ablates Zn 2+ release into the extracellular space by action potentials. There is a correlation between vesicular Zn 2+ levels and ZnT3 protein expression [bib_ref] ZnT-3, a putative transporter of zinc into synaptic vesicles, Palmiter [/bib_ref]. Zn 2+ transport into the synaptic vesicle is ZnT3-dependent and is important for amassing the large pool of Zn 2+ used in signaling [bib_ref] Dependence of the histofluorescently reactive zinc pool on zinc transporter-3 in the..., Lee [/bib_ref]. It has been reported that Zn 2+ signaling is involved in cognitive and emotional behavior even in ZnT3KO [bib_ref] Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic..., Adlard [/bib_ref] [bib_ref] Zinc transporter 3 is involved in learned fear and extinction, but not..., Martel [/bib_ref] [bib_ref] Zinc transporter 3 is involved in learned fear and extinction, but not..., Martel [/bib_ref] [bib_ref] Zinc transporter ZnT-3 regulates presynaptic Erk1/2 signaling and hippocampus-dependent memory, Sindreu [/bib_ref]. The pool of Zn 2+ may be located in other subcellular organelles and/or zinc-binding proteins such as metallothionein in ZnT3KO mice. On the other hand, memory deficit and the changes in emotional (freezing) behavior have been observed in wild-type animals when acute loss or chelation of synaptic Zn 2+ is induced by treatment with zinc chelators . The amount of Zn 2+ functioning as a signal factor seems to be lower in ZnT3KO mice than in wild-type mice. [bib_ref] Deficiencies of hippocampal Zn and ZnT3 accelerate brain aging of Rat, Saito [/bib_ref] report that age-dependent reduction of Zn 2+ levels in the synaptic vesicles of the mossy fibers induced by low ZnT3 expression causes both glutamatergic excitotoxicity in hippocampal neurons and the deterioration of learning and memory in senescence-accelerated mouse prone 10 (SAMP10). There are also reports of age-dependent reductions in ZnT3 expression and synaptic Zn 2+ levels in the hippocampal mossy fibers of human amyloid precursor protein-transgenic (Tg2576) mice, suggesting that extensive modifications of the brain's Zn 2+ pool, particularly synaptic (vesicular) Zn 2+ , underlie the neuronal dysfunction characteristic of Alzheimer's disease [bib_ref] Alteration of the cerebral zinc pool in a mouse model of Alzheimer..., Lee [/bib_ref]. Furthermore, there is a significant age-related decline in cortical ZnT3 levels from age 48 to 91 in healthy people [bib_ref] Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic..., Adlard [/bib_ref] and ZnT3 levels are more markedly decreased in the cortex in Alzheimer's disease. It is likely that the increase in extracellular Zn 2+ induced by the physiological excitation of zincergic neurons requires cognitive activity and that Frontiers in Aging Neuroscience www.frontiersin.org an insufficient increase is involved in the pathophysiology of Alzheimer's disease. On the other hand, Zn 2+ released from zincergic neurons is known to mediate parenchymal and cerebrovascular amyloid formation in Tg2576 mice [bib_ref] Contribution by synaptic zinc to the gender-disparate plaque formation in human Swedish..., Lee [/bib_ref] [bib_ref] Neuronal zinc exchange with the blood vessel wall promotes cerebral amyloid angiopathy..., Friedlich [/bib_ref] [bib_ref] Amyloid plaques arise from zinc-enriched cortical layers in APP/PS1 transgenic mice and..., Stoltenberg [/bib_ref]. The transsynaptic movement of Zn 2+ may be severely compromised in Alzheimer's disease, both by lack of ZnT3 expression and by sequestration in amyloid. [bib_ref] Cognitive loss in zinc transporter-3 knock-out mice: a phenocopy for the synaptic..., Adlard [/bib_ref] report that the genetic ablation of ZnT3 may represent a phenocopy for memory deficits in Alzheimer's disease. [bib_ref] A role for synaptic zinc in activity-dependent Abeta oligomer formation and accumulation..., Deshpande [/bib_ref] postulate that the sequestration of Zn 2+ in oligomeric amyloid-β (Aβ)-Zn complexes may lead to a reduction in Zn 2+ availability at the synapses, resulting in a loss of the modulatory activity of Zn 2+ , and leading to the cognitive decline of Alzheimer's disease. Such changes in synaptic Zn 2+ availability may participate in modifying cognitive activity and also in cognitive decline ; [bib_ref] The effects of enhanced zinc on spatial memory and plaque formation in..., Linkous [/bib_ref] [bib_ref] The metal theory of Alzheimer's disease, Bush [/bib_ref] [bib_ref] Altered expression of ZnT10 in Alzheimer's disease brain, Bosomworth [/bib_ref]. ## Glucocorticoid signaling, zn 2+ signaling, and cognition The hippocampus is enriched with corticosteroid receptors and is the major target region of corticosteroids [bib_ref] Functional actions of corticosteroids in the hippocampus, Joëls [/bib_ref]. Mineralocorticoid receptors and glucocorticoid receptors are colocalized in CA1 and CA2 pyramidal cells and in dentate gyrus granule cells. In CA3 pyramidal cells, on the other hand, mineralocorticoid receptors are abundantly expressed, while glucocorticoid receptors are expressed at much lower levels [bib_ref] Steroid hormones, their receptors and neuroendocrine system, Ozawa [/bib_ref]. Mineralocorticoid receptors are extensively occupied with low levels of corticosterone, and glucocorticoid receptors are particularly activated after exposure to stress [bib_ref] Glucocorticoids act on glutamatergic pathways to affect memory processes, Sandi [/bib_ref]. An increase in serum corticosterone level induces a rapid increase in hippocampal corticosterone level, in parallel with an increase in extracellular glutamate level [bib_ref] Rapid glucocorticoid effects on excitatory amino acid levels in the hippocampus: a..., Venero [/bib_ref]. Corticosterone-induced increase in extracellular glutamate levels in the hippocampus appears to be exerted through the action of membrane-associated mineralocorticoid receptors and/or glucocorticoid receptors, which increase the probability of glutamate release in synaptic activation [bib_ref] Mineralocorticoid receptors are indispensable for nongenomic modulation of hippocampal glutamate transmission by..., Karst [/bib_ref] [bib_ref] Acute stress increases depolarization-evoked glutamate release in the rat prefrontal/frontal cortex: the..., Musazzi [/bib_ref]. The rapid effects of corticosterone on glutamatergic transmission appear to be linked to diverse effects on synaptic plasticity and memory processes in the hippocampus . An increase in the probability of glutamate release through the action of corticosterone leads to increases both in the amount of glutamate released during learning and in the degree of activation of postsynaptic glutamate receptors. Corticosterone can contribute to an increase in the efficacy of glutamatergic transmission by AMPA receptor insertion at synaptic sites, through both the rapid and the delayed (genomic) effects. These effects are of advantage to processes of synaptic plasticity such as LTP and memory acquisition [bib_ref] Glucocorticoids act on glutamatergic pathways to affect memory processes, Sandi [/bib_ref]. Therefore, it is estimated that corticosterone increases the probability of Zn 2+ release from zincergic neuron terminals through the rapid non-genomic effect in the hippocampus ; . Futhermore, corticosterone requires intracellular Zn 2+ signaling for the genomic effect, possibly followed by the delayed influx of extracellular Zn 2+ through zinc transport systems such as ZIP . Although the evidence is limited, it is likely that synaptic Zn 2+ signaling cooperates with corticosteroid signaling in learning and memory. In contrast, glutamate accumulates in the extracellular compartment at high levels through a corticosterone-mediated blockade of glutamate transporter activity when corticosterone is abnormally secreted under conditions of severe stress. Abnormal corticosterone secretion also contributes to abnormal glutamate release from neuron terminals [bib_ref] Hippocampal long-term depression mediates acute stress-induced spatial memory retrieval impairment, Wong [/bib_ref] [bib_ref] Synaptic plasticity in learning and memory: stress effects in the hippocampus, Howland [/bib_ref]. The extracellular spillover of glutamate impairs spatial memory retrieval. Furthermore, [bib_ref] Hippocampal long-term depression mediates acute stress-induced spatial memory retrieval impairment, Wong [/bib_ref] demonstrate that hippocampal long-term depression (LTD) is both necessary and sufficient to cause acute stress-induced impairment of spatial memory retrieval. Excess intracellular Zn 2+ signaling induced by corticosterone and/or stress is also involved in the impairment of hippocampal LTP , possibly followed by the impairment of learning and memory . In hippocampal CA3, on the other hand, an increase in intracellular Zn 2+ via a zinc ionophore not only decreases basal Ca 2+ level but also suppresses increases in Ca 2+ level via metabotropic glutamate receptors [bib_ref] Role of zinc influx via AMPA/kainate receptor activation in metabotropic glutamate receptor-mediated..., Takeda [/bib_ref]. Such excess intracellular Zn 2+ signaling may lead to negative crosstalk in intracellular Ca 2+ signaling, which plays a key role in LTP and LTD . A selective increase in the nocturnal levels of corticol has been observed in aged humans [bib_ref] Evolving aspects of the glucocorticoid hypothesis of brain aging: hormonal modulation of..., Landfield [/bib_ref]. Furthermore, high levels of cortisol are found in Alzheimer's disease as well as in depression. In Alzheimer's disease patients, cognitive deficits (such as in memory) and psychological symptoms (such as anxiety) are associated with an early deregulation of the HPA axis [bib_ref] Hypothalamic-pituitary-adrenal axis dysfunction in Alzheimer's disease:lack of association between longitudinal and cross-sectional..., Swanwick [/bib_ref] [bib_ref] Deregulation of hypothalamic-pituitary-adrenal axis functions in an Alzheimer's disease rat model, Brureau [/bib_ref]. Therefore, it is possible that excess intracellular Zn 2+ signaling through abnormal cortisol secretion is involved in cognitive deficits in both normal aging and neurological diseases such as dementia. On the other hand, corticotrophin releasing hormone (CRH) drives the HPA axis and is considered to be the central coordinator of behavioral, autonomic, and neuroendocrine stress responses. The stress mediators activated by CRH are organized in the sympathetic nervous system, as well as in the HPA axis [bib_ref] Commentary: neuroendocrine basis. Prog, De Kloet [/bib_ref]. Adrenaline, along with norepinephrine, is largely responsible for the immediate reactions that are felt under conditions of stress. Responses of adrenaline and norepinephrine, such as an increase in heart rate, occur more quickly than those of glucocorticoids. Catecholamines are released from the sympathetic nerve system and the adrenal glad. It has been reported that the enhanced memory associated with emotional experiences involves activation of the β-adrenergic system [bib_ref] Beta-adrenergic activation and memory for emotional events, Cahill [/bib_ref] [bib_ref] Stress and cognitive function, Mcewen [/bib_ref]. β-Adrenergic receptor activation facilitates the induction of a protein synthesis-dependent late phase in LTP in the hippocampus [bib_ref] Beta-adrenergic receptor activation facilitates induction of a protein synthesis-dependent late phase of..., Gelinas [/bib_ref]. Learning-facilitated LTD and LTP at mossy fiber-CA3 synapses requires activation of βadrenergic receptors [bib_ref] Learning-facilitated long-term depression and long-term potentiation at mossy fiber-CA3 synapses requires activation..., Hagena [/bib_ref]. The above evidence suggests that synaptic Zn 2+ signaling is modified by the β-adrenergic system and is involved in cognitive activity associated with emotional experiences. The relationship between synaptic Zn 2+ signaling and the β-adrenergic system is an issue which requires further clarification. Stress is a known precipitant Frontiers in Aging Neuroscience www.frontiersin.org for metabolic and neurological diseases [bib_ref] Effects of stress across the lifespan, Koenig [/bib_ref] and synaptic Zn 2+ signaling is likely to be involved in the diverse effects of stress through the stress mediators activated by CRH. ## Perspective Synaptic Zn 2+ homeostasis is critical for synaptic function, and seems to be controlled by two Zn 2+ pools, one in the synaptic vesicle and the other in the extracellular compartment. Synaptic Zn 2+ signaling is involved in cognitive activity, and both its lack and its excess are involved in cognitive decline . HPA axis activity increases with aging, and this increase is superimposed on neurological diseases such as depression and Alzheimer's disease. It is likely that synaptic Zn 2+ signaling through the HPA axis activity is involved in cognitive decline in both normal aging and dementia, and it is possible that sympathetic nervous system activity is also involved. However, evidence related to synaptic Zn 2+ dynamics is very limited, not only under physiological conditions, but also under stressful and pathological conditions. The molecular mechanisms of abnormal Zn 2+ signaling in cognitive decline also remain to be clarified. # Acknowledgment We would like to thank Philip Hawke of the University of Shizuoka Scientific English Program for his comments on the English in the manuscript. February 2014 | Volume 6 | Article 26 | 1 February 2014 | Volume 6 | Article 26 | 2 February 2014 | Volume 6 | Article 26 | 3 February 2014 | Volume 6 | Article 26 | 4 February 2014 | Volume 6 | Article 26 | 5 February 2014 | Volume 6 | Article 26 | 6 February 2014 | Volume 6 | Article 26 | 7 Conflict of Interest Statement:The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

A new-onset pulmonary artery stenosis in a young man: case report # Introduction Pulmonary artery sarcoma is a rare type of malignant vascular tumour characterized by a very aggressive clinical course and an unfavourable prognosis.Its rarity and the absence of specific symptoms, especially at the onset, make the diagnosis difficult and often delayed, or only post-mortem.The therapeutic options are limited and often suboptimal, and the median survival without radical resection is about CASE REPORT 30-50 days.In this report, we present the case of a young man with an isolated pulmonary artery sarcoma. ## Timeline case presentation A 31-year-old, non-smoking male presented to the hospital complaining of progressive asthenia and exertional dyspnoea for 4 months before. He had an unremarkable past medical history and physical examination. Before the patient presented at hospital, the spirometry had provided normal results, a high resolution computed tomography (HRCT) pulmonary scan had shown a non-specific inflammatory parenchymal pattern and an enlarged pulmonary artery trunk. In addition, echocardiography showed a marked increase of transpulmonary peak flow velocity and an unspecified mass in the pulmonary arterial trunk. The patient was admitted to the hospital for further investigation. On admission, his vital signs were stable, and peripheral oxygen saturation was normal. Laboratory data showed mildly increased inflammatory markers (D-dimer: 528 lg/L, fibrinogen: 609 mg/dL, high-sensitivity C-reactive protein (hs-CRP): 23 mg/L) and low ferritin, while white blood cell count, haemoglobin, renal function, hepatic and cardiac enzymes were normal. The laboratory investigation for genetic thrombophilia was negative. Echocardiographyconfirmed a mass with irregular borders within the pulmonary trunk, almost obliterating its lumen and determining a flow acceleration with a peak velocity and a gradient of 3.8 m/s and 60 mmHg, respectively. All other Doppler, morphologic and functional findings of the pulmonary valve and right ventricle were normal. Initially, these findings were interpreted as possible venous thromboembolism, and we started anticoagulant therapy with low molecular weight heparin. However, compression ultrasonography ruled out a deep vein thrombosis. Repeated pulmonary HRCT showed dilatation of the pulmonary trunk with an irregular mass within its lumen with extremely limited enhancement by contrast medium, mistakenly reported as a big thrombus, and isolated areas of ground-glass opacity in the lung parenchyma for which we started large spectrum antibiotic therapy. Colonoscopy and esophagogastroduodenoscopy were performed to exclude a paraneoplastic thrombosis and were both negative for malignancies. Follow-up echocardiography performed after 10 days demonstrated a dilated right ventricle with increased systolic pressure. Due to the atypical clinical manifestations and the unresponsiveness to anticoagulant therapy with Low-molecular-weight heparin (LMWH), we performed a cardiac magnetic resonance (MR)that confirmed an intravascular mass with a thrombotic component adherent to the right side of the pulmonary trunk and inhomogeneous impregnation by contrast medium, highly suggestive for an angiosarcoma of the pulmonary artery. Positron emission tomography scan (PET-SCAN) with 18-fluorodeoxyglucose ( 18 F-FDG)revealed an intense local glucose metabolism at the pulmonary trunk level, without any extrapulmonary lesion, which excluded a metastatic nature of the mass. Finally, the lesion was biopsied by bronchoscopy transbronchial, but the resulting material was inadequate for a specific diagnosis. The histologic report highlighted the presence of rare and isolated atypic cells, not further typable, interspersed with cylindric hair cells and globular red cells; it was technically impossible to search for the different types of markers, with the exception of Citokeratyn 5.2 (CAM 5.2), which was positive. The patient decided to continue the treatment in another hospital, where he was operated 2 weeks after diagnosis. However, he developed a local relapse after 6 months and died within a few weeks. ## Data Events and diagnostic tests ## Learning points - The diagnosis of artery sarcoma must be suspected in patients with relevant respiratory symptoms but a low probability of pulmonary embolism (e.g. Wells score < _1). - The evaluation of an intravascular mass requires a multiparametric approach for better tissue characterization and assessment of its haemodynamic impact. - No guidelines are currently available. Therapeutic strategy must be individually tailored, combining surgical and medical treatment. Radical resection seems to be the mainstay of therapy. . # Discussion Primary pulmonary artery sarcomas are rare malignant tumours whose proliferative cells arise from the mesenchymal cells of the pulmonary artery.It is the most common primary tumour of the pulmonary arteries, usually affecting large-calibre branches with predominantly intraluminal growth.There are some different histopathologic patterns of primary artery sarcomas (undifferentiated sarcoma, rhabdomyosarcoma, leiomyosarcoma, spindle cells sarcoma, angiosarcoma, and few other rare forms), but the histopathological classification does not seem to have clinical or prognostic usefulness.These tumours may metastasize to the lung, brain, liver, spleen, small bowel, and adrenal glands.Bandyopadhyay et al.,in their literature research from 1991 to 2010, reported 391 primary pulmonary artery sarcomas; the first published case was in 1923 from an autopsy by Mandelstam.Symptoms of pulmonary sarcoma are non-specific: dyspnoea, cough, chest pain, malaise, haemoptysis, pulmonary hypertension, and lipothymic events, due to right ventricular dysfunction or pulmonary valve obstruction, are frequently reported. 1,2,4 As symptoms are non-specific, the diagnosis is often delayed. Indeed, the tumour may develop silently, and symptoms of pulmonary obstruction may be the first manifestation, usually at an advanced stage.The differential diagnosis includes lung cancer, pulmonary embolism, pulmonary hypertension, and congenital pulmonary stenosis when the pulmonary valve is involved,Pulmonary embolism is the most frequent misdiagnosis.Pulmonary artery sarcoma should be suspected whenever there is no clinical improvement with anticoagulant therapy in patients with a low probability of thromboembolic events (Wells clinical prediction score < _1).Clinical and laboratory data may help in the differential diagnosis: fever, high hs-CRP, or Erythrocyte Sedimentation Rate (ESR), weight loss, lack of history of deep vein thrombosis, absence of procoagulant mutations, should all orient towards a non-thrombotic cause.Chest radiography and computed tomography (CT)-scans may show different patterns, from solitary lesions to multiple nodular densities, with or without pleural effusion. Multiple infiltration or consolidation and metastatic localization suggest an unfavourable prognosis short term. Thanks to gadolinium-enhancement, magnetic resonance imaging increases the diagnostic sensitivity, while intense 18 F-FDG uptake at PET helps to differentiate sarcoma from thrombi.The different histologic types of sarcomas are frequently indistinguishable at radiologic analysis, but morphological features, location, and clinical presentation may guide in the diagnosis.However, definitive diagnosis needs histopathological examination with immunohistochemical markers including desmin, cytokeratin, vimentin, and actin. CD 31, CD34, and factor VIII-related antigen are more specific for angiosarcoma.Also because of its rarity, guidelines for the management of pulmonary sarcoma are not consolidated. Surgical resection, combined with chemo-radiotherapy and immunotherapy, seems to offer the best survival rates 1,2,4,6 ; nevertheless, the tumour is very aggressive, and the prognosis remains poor, particularly if a complete resection is not feasible, as it happens when the tumour is not locally confined.Some anti-tumoural agents like anthracycline (doxorubicin, epirubicin) with or without an alkilant (dacarbazine, ifosfamide) have been tested. Other agents are vincristine, paclitaxel, gemcitabine, etoposide, interleukin-2, vincristine, mitomycin, methotrexate, and sunitinib.Radiotherapy may be used post-surgery to avoid local relapse.Bandyopadhyay et al.in their retrospective observational analysis, comprehensive of their hospital cases and cases from the literature, showed that adjunctive therapy (chemo/radiotherapy) improves survival and reduces development of distant metastasis but not local recurrence. The improvement of survival with multimodality therapy was also confirmed by Blackmon et al.Neoadjuvant chemotherapy has been described in stable patients and when the patients were not candidate for a curative resection; Linden et al. reported a case whereby tumour downstaging with neoadjuvant chemotherapy allowed a radical surgery to be performed.Furthermore, the review conducted by Blackmon et al.on the treatment of pulmonary artery sarcomas combined between literature cases and theirs shows that a curative resection attempt improves the median and 5 years of survival compared with debulking or palliative surgery. The possibility of a radical resection remains the most important prognostic factor, which is more likely with an early diagnosis, and with a high definition by multimodal imaging. 1,2,6 Therefore, radical

Laparoscopic Colorectal Resection in Octogenarian Patients The population older than 80 years has been increasing. A significant proportion of colorectal diseases that require colorectal resection occur in very elderly patients. However, the benefits of laparoscopy remain controversial in octogenarians. A systematic review and meta-analysis of observational study was performed to compare clinical outcomes between laparoscopic versus open colorectal resection in octogenarians.The PubMed, EMBASE, Ovid, Web of Science, and Cochrane databases from the years 1990 to 2015 were searched for studies that compare surgical outcomes between laparoscopic and open colorectal resection in octogenarians (!80 years old).Seven eligible studies including 528 laparoscopic and 484 open colorectal resections were identified. Laparoscopic approach was associated with lower rate of mortality (odds ratio [OR] 0.48, P ¼ 0.03), overall complications (OR 0.54, P < 0.001), and prolonged ileus (OR 0.56, P ¼ 0.009), quicker bowel function return (standardized mean difference [SMD] À0.50, P < 0.001), and shorter length of hospital stay (SMD À0.47, P ¼ 0.007). No differences were found in anastomotic leak (OR 1.16, P ¼ 0.72), respiratory complication (OR 0.60, P ¼ 0.07), and reoperation (OR 0.85, P ¼ 0.69).Laparoscopic colorectal resection is as safe as open approach, and the short-term outcomes appear to be more favorable in octogenarians. (Medicine 94(42):e1765) Abbreviations: CI = confidence interval, LC = laparoscopic colorectal resection, OC = open colorectal resection, OR = odds ratio, SMD = standardized mean difference.Editor:à Random-effect model was adopted. y Indicates odds ratio. z Indicates standardized mean difference. FIGURE 2. Funnel plot of included studies.FIGURE 3. Pooled data on safety outcomes between laparoscopic and open colorectal resection in octogenarians. A, Forest plot of overall complication. B, Forest plot of wound infection. C, Forest plot of prolonged ileus. D, Forest plot of cardiovascular complication. E, Forest plot of mortality. LC ¼ laparoscopic colorectal resection, OC ¼ open colorectal resection, OR ¼ odds ratio. # Introduction W ith the rapid improvement in health care and advances in modern medicine, global life expectancy increased dramatically over the past decade, and more people are expected to live to an advanced age. It is estimated that half of the individuals born today will live to the age of 100 years. [bib_ref] Ageing populations: the challenges ahead, Christensen [/bib_ref] In the United States, the population older than 80 years has increased from 9.19 million (3.26%) to 11.28 million (3.64%) over the past decade.Along with this is the increase in the number of very old patients. A significant proportion of colorectal diseases that require colorectal resection occur in this patient group. An article published in the Lancet 3 showed that the incidence of postoperative morbidity and mortality increased progressively with age, and the overall survival reduced in the older patients, especially in the patients aged 65 years or older. This study also found that comorbidities, especially pulmonary diseases and cardiovascular problems in the older patients, were associated with higher incidence of postoperative morbidity and mortality. Some randomized controlled trials have shown that laparoscopic colorectal surgery is safe and is associated with definite benefits in short-term surgical outcomes, such as shorter length of hospital stay, less postoperative pain, earlier recovery of bowel function, and more rapid return to regular activities, compared with conventional open surgery for selected adult patients. [bib_ref] Laparoscopyassisted colectomy versus open colectomy for treatment of nonmetastatic colon cancer: a..., Lacy [/bib_ref] [bib_ref] COLOR: a randomized clinical trial comparing laparoscopic and open resection for colon..., Hazebroek [/bib_ref] It is proposed that laparoscopic surgery may be the ideal option for the elderly patients. [bib_ref] Laparoscopy for benign colorectal diseases, Shin [/bib_ref] However, laparoscopic approach is associated with specific physiologic changes, which might influence the older patients adversely. For instance, the Trendelenburg positioning during laparoscopy and pneumoperitoneum may result in a significant reduction in stroke volume and cardiac outputs. [bib_ref] Diastolic function: the influence of pneumoperitoneum and Trendelenburg positioning during laparoscopic hysterectomy, Russo [/bib_ref] These factors, together with existing comorbidities, have made surgeons reluctant to offer laparoscopy to the older patients. [bib_ref] Laparoscopic resection of rectosigmoid carcinoma: prospective randomised trial, Leung [/bib_ref] [bib_ref] Safety and advantages of laparoscopic vs. open colectomy in the elderly: matched-control..., Stocchi [/bib_ref] However, Chautard et al 10 demonstrated, in a large case-matched study, that laparoscopic colorectal resection (LC) in older patients (>70 years) is similar to that in the younger patients (<70 years) in terms of early postoperative outcomes, despite significant higher incidence of cardiopulmonary comorbidities in the senior cohort. Therefore, we hypothesized that LC confers benefits over the open colorectal resection (OC) for the older population, particularly those who are aged 80 years or above. The present meta-analysis was performed in an attempt to evaluate the safety and efficacy between LC versus OC in octogenarians. # Methods ## Literature search A literature search of the PubMed, EMBASE, Ovid, Web of Science, and Cochrane databases from the years 1990 to 2015 was performed to identify articles comparing laparoscopic versus open surgery for octogenarians undergoing colorectal resection for cancer or other diseases. The following search terms and their combinations were used: (laparoscop à OR minimally invasive), (colectomy OR colorectal resection/surgery), (octogenarian à OR elderly OR older than 80). Both free text search and Mesh search headings for keywords were employed. The ''related articles'' function was used to broaden the search, and relevant articles referenced in the publications were also searched for additional studies for potential inclusion. No language restriction was used. The date of the most recent search was March 20, 2015. ## Study selection For inclusion in the meta-analysis, studies were required to fulfill the following criteria: clear definition of surgical approach (laparoscopic vs open colorectal surgery); appropriate age cut-off (aged 80 or above); concurrent controls included; adequate data to determine OR and confidence intervals (CIs); and no later publication of identical data. Noncomparative studies, case series, and case reports were not included. Two authors (MX and HQ) independently assessed titles and abstracts of all identified studies and excluded those deemed irrelevant. Full-text articles of potentially relevant studies were obtained. Disagreements on inclusion were discussed and, if necessary, by involving an independent third author (LL). ## Outcome measurements Safety outcomes included total complication rate, wound infection, anastomotic leak, prolonged ileus, cardiovascular complication, respiratory complication, and mortality. Surgical outcomes included bowel function recovery time, length of hospital stay, and perioperative reoperation. ## Quality and bias assessment The quality of all the included studies was assessed by 2 authors (MX and HQ) using the modified Newcastle-Ottawa Scale.The quality of the studies was evaluated by examining 3 items: patient selection, comparability of LC and OC groups, and assessment of outcome. Graphical exploration with funnel plots was used to evaluate publication bias. [bib_ref] Bias in meta-analysis detected by a simple, graphical test, Egger [/bib_ref] # Statistical analysis Odds ratios were used to represent the likelihood of an adverse event in the LC group compared with that in the OC group. The ORs were combined using the Mantel-Haenszel method for the outcomes of interest. [bib_ref] Statistical aspects of the analysis of data from retrospective studies of disease, Mantel [/bib_ref] Statistical analyses for continuous variables were performed using the standardized mean difference (SMD). A negative SMD favored the laparoscopic surgery group. For all outcomes, the fixed and random-effect models were calculated. When I 2 test rejected the assumption of study homogeneity (I 2 value >50%), the fixed-effect model was considered inappropriate, and the random-effect model was adopted. For continuous variables such as medians with ranges, the mean and standard deviation were estimated as previously described by Hozo et al. 14 All analyses were conducted using R (version 3.1.3, R Foundation for Statistical Computing, Vienna, Austria) with the ''meta'' package. A P value <0.05 was considered statistically significant. ## Ethics approval The study was reviewed and approved by the Institutional Review Board and the Ethics Committee of the Sixth Affiliated Hospital of Sun Yat-sen University, Guangzhou, China. # Results The inclusion and exclusion processes for identified articles are shown in . Seven observational studies were finally included in the meta-analysis, comparing laparoscopic versus open surgery for octogenarians undergoing colorectal resection, using the predefined search strategy, containing a total of 1012 patients. There was no randomized controlled trial published. All the studies were single institutional. Sample size of the studies varied from 77 to 245 patients. Review of the data extraction showed 100% agreement between the 2 reviewers. There were 528 (52.2%) patients in the LC group and 484 (47.8%) patients in the OC group. The characteristics of the included studies are shown in [fig_ref] TABLE 1: Characteristics of Included Controlled Studies [/fig_ref]. Surgery-related details are shown in [fig_ref] TABLE 2: Surgery-related Details of Included Studies [/fig_ref]. No significant differences were found between the 2 groups in indication for surgery or type of procedure in any of these studies. From the quality assessment based on the Newcastle-Ottawa Scale, all the studies included were of good to high quality (!6 stars). As shown in , the funnel plot revealed no publication bias of the included studies (P ¼ 0.65). Eleven measurement indices comparing LC versus OC were concerned, which were listed in [fig_ref] TABLE 3: Meta-analysis of the Outcomes After Laparoscopic and Open Colectomyrange in some studies,... [/fig_ref] after pooled analysis. ## Safety outcomes ## Overall complication Five studies [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopic colectomy for carcinoma of the colon in octogenarians, Issa [/bib_ref] [bib_ref] Laparoscopic colectomy in octogenarians and nonagenarians: a preferable option to open surgery?, Kurian [/bib_ref] assessed occurrences of at least 1 complication . The total complication rates were recorded. A pooled analysis combining the effects of 5 studies showed that the overall complication rate of LC group in the octogenarians was significantly lower than that of the OC group (28.1% vs 42.9%; OR 0.54, 95% CI 0.40-0.71, P < 0.001). ## Wound infection Laparoscopic surgery in the octogenarians has a reportedly lower wound infection rate in all 7 studies 15-21 ; however, the difference was statistically significant in only one of them. [bib_ref] Laparoscopically assisted colorectal surgery in the elderly, Stewart [/bib_ref] Overall, among the 1012 analyzed patients, LC was associated with a significantly lower incidence of wound infection rate when compared with OC (OR 0.51, 95% CI 0.32-0.81, P ¼ 0.005). ## Anastomotic leak Anastomotic leak was investigated in 6 studies, [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopic colectomy for carcinoma of the colon in octogenarians, Issa [/bib_ref] [bib_ref] Laparoscopically assisted colorectal surgery in the elderly, Stewart [/bib_ref] [bib_ref] Laparoscopic resection for colorectal cancer improves short-term outcomes in very elderly colorectal..., Hatakeyama [/bib_ref] which showed no significant difference between the LC and the OC groups in octogenarians. After pooling the effects, the difference remained statistically insignificant (OR 1.16, 95% CI 0.52-2.58, P ¼ 0.72). ## Prolonged ileus The prolonged ileus rate was reported in 5 studies including 837 patients . [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopically assisted colorectal surgery in the elderly, Stewart [/bib_ref] Only 1 study found statistical difference. [bib_ref] Laparoscopic colectomy in octogenarians and nonagenarians: a preferable option to open surgery?, Kurian [/bib_ref] After pooling the effects, LC was associated with a significantly lower incidence of prolonged ileus rate when compared with OC (OR 0.56, 95% CI 0.36-0.86, P ¼ 0.009). ## Cardiovascular complication Data on cardiovascular complication were reported in 6 studies including 919 patients, which showed lower cardiovascular complication rate in the laparoscopic surgery group . Only 1 study found statistical difference. [bib_ref] Laparoscopically assisted colorectal surgery in the elderly, Stewart [/bib_ref] By pooling the data, it was found that LC was associated with lower cardiovascular complication rate (OR 0.60, 95% CI 0.36-0.99, P ¼ 0.04). ## Respiratory complication Pooled analysis of respiratory complication across 6 studies [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopic colectomy for carcinoma of the colon in octogenarians, Issa [/bib_ref] [bib_ref] Laparoscopically assisted colorectal surgery in the elderly, Stewart [/bib_ref] [bib_ref] Laparoscopic resection for colorectal cancer improves short-term outcomes in very elderly colorectal..., Hatakeyama [/bib_ref] noted that there was no difference between the 2 treatment groups (OR 0.60, 95% CI 0.34-1.05, P ¼ 0.07). ## Mortality Postoperative mortality here was defined as the death within 30 days of surgery. Pooled data from all 7 studies [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopic colectomy for carcinoma of the colon in octogenarians, Issa [/bib_ref] [bib_ref] Laparoscopic colectomy in octogenarians and nonagenarians: a preferable option to open surgery?, Kurian [/bib_ref] [bib_ref] Laparoscopically assisted colorectal surgery in the elderly, Stewart [/bib_ref] [bib_ref] Laparoscopic resection for colorectal cancer improves short-term outcomes in very elderly colorectal..., Hatakeyama [/bib_ref] showed that LC was associated with a significantly lower mortality when compared with OC (OR 0.48, 95% CI 0.25-0.92, P ¼ 0.03) . ## Surgical outcomes ## Bowel function return Data on bowel function return were available in 5 studies [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopic colectomy for carcinoma of the colon in octogenarians, Issa [/bib_ref] [bib_ref] Laparoscopic resection for colorectal cancer improves short-term outcomes in very elderly colorectal..., Hatakeyama [/bib_ref] including 690 patients. The bowel function return was defined by oral intake initiated after the first flatus. The data were recorded starting on the morning after surgery for 24 h. Pooled analysis showed a shorter recovery time in the laparoscopic group (OR À0.50, 95% CI À0.65 to À0.34, P < 0.001) . ## Length of hospital stay Issa et al [bib_ref] Laparoscopic colectomy for carcinoma of the colon in octogenarians, Issa [/bib_ref] reported postoperative hospital stay, whereas the other 6 studies concerned overall hospital stay. We conducted overall hospital stay by pooling the 6 studies, [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopic colectomy in octogenarians and nonagenarians: a preferable option to open surgery?, Kurian [/bib_ref] [bib_ref] Laparoscopically assisted colorectal surgery in the elderly, Stewart [/bib_ref] [bib_ref] Laparoscopic resection for colorectal cancer improves short-term outcomes in very elderly colorectal..., Hatakeyama [/bib_ref] showing a favorable heterogeneity (I 2 ¼ 83.7%). The randomeffect model was used, which suggested that laparoscopic approach was associated with a shorter hospital stay (SMD À0.47, 95% CI À0.81 to À0.13, P ¼ 0.007) . ## Reoperation Four studies [bib_ref] Laparoscopic vs. open colectomies in octogenarians: a case-matched control study, Vignali [/bib_ref] [bib_ref] Laparoscopic colectomy is safe and leads to a significantly shorter hospital stay..., Lian [/bib_ref] [bib_ref] How reliable is laparoscopic colorectal surgery compared with laparotomy for octogenarians?, Pinto [/bib_ref] [bib_ref] Laparoscopic colectomy for carcinoma of the colon in octogenarians, Issa [/bib_ref] including 608 patients reported the occurrence of reoperation. There was no significant difference found between the 2 groups in all the 4 studies. The pooled OR was comparable (OR 0.85, 95% CI 0.39-1.85, P ¼ 0.70). # Discussion This systematic review and meta-analysis of observational studies was performed to compare clinical outcomes between LC versus OC in octogenarians. The 7 included observational studies available so far suggest that laparoscopic approach was associated with lower rate of prolonged ileus, mortality, and overall complications, as well as quicker bowel function return and shorter length of hospital stay. The short-term outcomes of laparoscopic colorectal resection seem to be more favorable in octogenarians. Over the past decade, laparoscopy colorectal resection is now accepted as not only feasible but also advantageous. [bib_ref] Laparoscopic resection of rectosigmoid carcinoma: prospective randomised trial, Leung [/bib_ref] [bib_ref] Safety and advantages of laparoscopic vs. open colectomy in the elderly: matched-control..., Stocchi [/bib_ref] [bib_ref] Meta-analysis of short-term outcomes after laparoscopic resection for colorectal cancer, Abraham [/bib_ref] Previous studies have shown that colorectal surgery in older patients is generally well tolerated as compared to that in younger patients. [bib_ref] Laparoscopic colorectal surgery in elderly patients: a matched case-control study in 178..., Chautard [/bib_ref] [bib_ref] Age and type of procedure influence the choice of patients for laparoscopic..., Sklow [/bib_ref] The surgical outcomes of LC in octogenarians have been previously studied with conflicting results. Compared with open surgery, laparoscopic colorectal resection demonstrated better short-term outcomes in several published [bib_ref] Laparoscopic resection for colorectal cancer improves short-term outcomes in very elderly colorectal..., Hatakeyama [/bib_ref] All cancer NA randomized clinical trials. [bib_ref] COLOR: a randomized clinical trial comparing laparoscopic and open resection for colon..., Hazebroek [/bib_ref] [bib_ref] Diastolic function: the influence of pneumoperitoneum and Trendelenburg positioning during laparoscopic hysterectomy, Russo [/bib_ref] [bib_ref] Laparoscopic resection of rectosigmoid carcinoma: prospective randomised trial, Leung [/bib_ref] However, older patients with significant comorbidities were excluded in the majority of these clinical trials. Therefore, more data are needed to confirm the potential benefits of LC in the octogenarians. The hemodynamic and ventilation changes observed in laparoscopic surgery were considered as concerning factors for the octogenarians.On the contrary, the minimal invasive nature of laparoscopy may minimize surgical stress onto the older patients. Possible benefits of laparoscopy in this age group remain unclear. Therefore, the adoption of laparoscopy in octogenarians has not been a common practice. This study aimed to compare the safety and surgical outcomes of LC with OC for the octogenarians. The results of this meta-analysis suggest that laparoscopic resection is advantageous in most of the analyzed outcomes for colorectal diseases in octogenarians. Lower overall complication rate and less wound complication are shown as observed in other age groups. [bib_ref] Safety and advantages of laparoscopic vs. open colectomy in the elderly: matched-control..., Stocchi [/bib_ref] [bib_ref] Meta-analysis of short-term outcomes after laparoscopic resection for colorectal cancer, Abraham [/bib_ref] Although longer operation time and the Trendelenburg position are usually required during a laparoscopic procedure, fewer cardiovascular complication and similar respiratory complication suggest that the hemodynamic and ventilation change are well tolerated and laparoscopic technique could be safely used in older patients. This is in accordance with the study by Zhu et al 25 which showed that with cautious perioperative monitoring, carbon dioxide pneumoperitoneum is safe for the older patients without significant adverse effects. The benefits brought by the less surgical operation and stress prevailed over the disadvantages in laparoscopy mentioned above in the octogenarians. Fielding et al [bib_ref] Factors influencing mortality after curative resection for large bowel cancer in elderly..., Fielding [/bib_ref] reported that pulmonary complications are among the most significant problems in the postoperative period after colorectal resections, and cardiopulmonary complications after surgery in older patients are a major cause of postoperative morbidity and mortality. Lower overall complications perhaps contributed to the lower mortality between the 2 procedures in this study. About surgery-related complications, there were no differences in anastomotic leak and lower incidence of prolonged ileus. All complications mentioned above suggest that LC in the octogenarians is at least as safe as open surgery. Length of hospital stay in octogenarians after LC has been shown to be shorter than that of the OC group. A newly published retrospective literature by White et al [bib_ref] A comparison of laparoscopic versus open rectal surgery in 114 consecutive octogenarians, White [/bib_ref] found similar results on postoperative complications and length of hospital stay of LC versus OC in octogenarians. However, necessary data were not provided in this study, so it was not included in this meta-analysis. We also found a quicker bowel function return for the laparoscopic group. These results are in accordance with results of several other published reports, indicating that patients in other age groups undergoing LC have quicker postoperative recovery when compared to OC. 9,28 These findings suggest that the overall cost may be decreased when laparoscopic approach is used for octogenarians, which cannot be determined because of unavailability of the pertinent data. The present study has inherent limitations. Firstly, all the included studies were retrospective design with selection bias. Larger sample and better-designed studies are needed to verify our conclusions. Secondly, the patient characteristics, surgical protocols, surgeon experience, and postoperative care regimens are not unified among the included studies and sometimes not available. For example, data were recorded by the median and # Conclusions Laparoscopic colorectal resection is as safe as open approach, and the short-term outcomes seem to be more favorable in octogenarians. However, clinical value of LC in octogenarians needed further evaluation with better-designed and long-term follow-up studies. [table] TABLE 1: Characteristics of Included Controlled Studies [/table] [table] TABLE 2: Surgery-related Details of Included Studies [/table] [table] TABLE 3: Meta-analysis of the Outcomes After Laparoscopic and Open Colectomyrange in some studies, but others used mean and standard deviation. Therefore, bias might not be obviated. [/table]

The spectrum of genetic variants and phenotypic features of Southeast Asian patients with Noonan syndrome Background: Noonan syndrome (NS) is an autosomal dominant disorder that belongs to a group of developmental disorders called RASopathies with overlapping features and multiple causative genes. The aim of the study was to identify mutations underlying this disorder in patients from Southeast Asia and characterize their clinical presentations. Methods: Patients were identified from the hospital's Genetics clinics after assessment by attending clinical geneticists. A targeted gene panel was used for next-generation sequencing on genomic DNA extracted from the blood samples of 17 patients. Results: Heterozygous missense variants were identified in 13 patients: eight were in PTPN11, three in SOS1, and one each in RIT1 and KRAS. All are known variants that have been reported in patients with NS. Of the 13 patients with identified variants, 10 had short stature, the most common feature for NS. Four of the eight patients with PTPN11 variants had atrial septal defect. Only two had pulmonary stenosis which is reported to be common for PTPN11 mutation carriers. Another two had hypertrophic cardiomyopathy, a feature which is negatively associated with PTPN11 mutations. Conclusions: Our study provides the mutation and phenotypic spectrum of NS from a new population group. The molecular testing yield of 76% is similar to other studies and shows that the targeted panel approach is useful for identifying genetic mutations in NS which has multiple causative genes. The molecular basis for the phenotypes of the remaining patients remains unknown and would need to be uncovered via sequencing of additional genes or other investigative methods.K E Y W O R D Sgenetic testing, molecular diagnosis, Noonan syndrome, RASopathies 2 of 9 | KOH et al. # | introduction Noonan syndrome is an autosomal dominant disorder with variable expressivity. It is one of the more common Mendelian disorders with the reported frequency of one in 1,000-2,500 live births. There has been no recent update on the prevalence observed, and also no country-or population-specific data. Noonan syndrome is caused by germline mutations in genes encoding components belonging to the Ras and mitogen-activated protein kinase (MAPK) signaling pathway that interacts with extracellular growth factors to regulate cell proliferation, differentiation, and senescence. Although somatic mutations of Ras/MAPK pathway genes are found in many cancers, germline mutations are behind a group of developmental disorders whose presentations include craniofacial dysmorphism, short stature, congenital heart disease, and skeletal abnormalities. The disorders are collectively called RASopathies, among which NS is the most common. RASopathies also include Noonan syndrome with multiple lentigines (NSML) which was previously known as LEOPARD syndrome (OMIM 151100), Costello syndrome (OMIM 218040), cardiofaciocutaneous (CFC) syndrome , other Noonan-like syndrome (OMIM 607721), Legius syndrome (OMIM 611431), and neurofibromatosis type 1 (OMIM 162200). As these syndromes have overlapping clinical features with variable expressivity and some manifestations are also age-dependent, establishing the correct diagnosis can be challenging. The variable clinical phenotype behind NS and Noonanlike disorders is partly due to genetic heterogeneity, where mutations in multiple genes can result in different phenotypic expressions. To date, mutations in PTPN11 (OMIM 176876), SOS1 (OMIM 182530), RAF1 (OMIM 164760), KRAS , NRAS (OMIM 164790), BRAF (OMIM 164757), RIT1 (OMIM 609591), SOS2 (OMIM 601247), and LZTR1 (OMIM 600574) have been identified in patients with NS, with PTPN11 accounting for 50%-60% of identified mutations [bib_ref] Noonan syndrome-causing genes: Molecular update and an assessment of the, El Bouchikhi [/bib_ref] [bib_ref] The RASopathies, Rauen [/bib_ref] [bib_ref] Noonan syndrome -a new survey, Tafazoli [/bib_ref]. For Costello syndrome, mutations in HRAS (OMIM 190020) have been identified in 83%-100% of patients and is the only gene definitely associated with the syndrome thus far . PTPN11, RAF1, BRAF, and MAP2K1 (OMIM 176872) are mutated for NSML; while BRAF, KRAS, MAP2K1, and MAP2K2 (OMIM 601263) are causative genes for CFC syndrome [bib_ref] Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy, Pandit [/bib_ref] genes have mutations identified in more than one related disorders, such as PTPN11 for both NS and NSML [bib_ref] Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy, Pandit [/bib_ref] [bib_ref] Noonan syndrome: Clinical aspects and molecular pathogenesis, Tartaglia [/bib_ref] , and KRAS for NS and CFC , MAP2K1 and SOS1 for CFC and NS [bib_ref] Genotype and phenotype in patients with Noonan syndrome and a RIT1 mutation, Kouz [/bib_ref]. For these genes, the resulting disorder would usually depend on whether the specific disease-causing variant results in gain or loss of function of the encoded protein. As mutations in different genes have been found in patients with the different disorders, identifying the causative gene mutation would help to differentiate Noonan from Noonan-like syndromes and other RASopathies. Since different disorders have different prognosis, a molecular confirmation of the clinical diagnosis will result in more targeted medical management to improve the patient's long-term outcome and quality of life. For example, specific cardiac follow-up is recommended for hypertrophic cardiomyopathy (HCM) in NS and Costello syndrome [bib_ref] Clinical presentation and natural history of hypertrophic cardiomyopathy in RASopathies, Calcagni [/bib_ref]. On the other hand, patients with syndromes that have higher risk for malignancy (such as Costello syndrome) require disease-specific tumor surveillance. The spectrum of mutations for NS has been reported in European and American populations, and also several countries in Western and Northern Asia. However, the mutation spectrum has not been characterized in patients from Singapore. In this study, we utilized next-generation sequencing to identify the genetic mutations and evaluate the phenotypic features in our NS patients on genetic follow-up in Singapore, where >95% of the country's population are of Chinese, Malay, or Indian descent. ## | materials and methods ## | ethical compliance and enrollment of subjects The study was protocol was reviewed and approved by the SingHealth Centralized Institutional Review Board, Singapore. A written informed consent was obtained from all subjects' parents. Patients with clinical suspicion of NS from the Genetics clinics in KK Women's and Children's Hospital were assessed by the attending clinical geneticists, using the criteria developed by as a guide. ## | dna sequencing Genomic DNA was extracted from venous blood and sequenced using a custom-designed targeted panel that included all exons and exon-intron junctions of PTPN11, NF1, BRAF, KRAS, NRAS, HRAS, SOS1, RAF1, SHOC2, CBL, RIT, MAP2K1, MAP2K2, SPRED1, and RASA1. The DNA samples were prepared for sequencing using the HaloPlex TM Target Enrichment System (Agilent Technologies, Santa | 3 of 9 KOH et al. Clara, CA) and sequenced on the MiSeq System (Illumina, San Diego, CA). Data were processed using the MiSeq Reporter pipeline and annotated using wANNOVAR [bib_ref] wANNOVAR: Annotating genetic variants for personal genomes via the web, Chang [/bib_ref]. Sequence variants were checked against those available in dbSNP, 1,000 Genomes, ExAC, ClinVar, and Human Genome Mutation Database. Variants not previously reported in population databases or not previously classified as pathogenic were evaluated using SIFT and Polyphen predictions. All identified variants were confirmed via Sanger sequencing. Targeted Sanger sequencing of parental samples for variants identified in the patients was used to establish inheritance. Information on frequencies in population databases, previous reports in patients with similar phenotypes, and inheritance status were used to classify the pathogenicity of the variants according to the guidelines by ClinGen's RASopathy Expert Panel [bib_ref] ClinGen's RASopathy Expert Panel consensus methods for variant interpretation, Gelb [/bib_ref]. # | results ## | study subjects Seventeen unrelated patients were enrolled into the study from July 2014 to November 2015. The demographic characteristics of the patients are presented in . Nine patients were already on follow-up with our hospital before study commencement, the remaining eight were recruited during their first consultation with the Genetics Service clinics. The median age at clinical assessment was 3 years old. ## | clinical evaluation Complete data on all 17 patients wer only available for eight clinical features , of which the most common was short stature (12/17 [70.6%]), followed by pectus deformity . For clinical features with incomplete data, the most prevalent was cardiac abnormality (10/16) with pulmonary stenosis (PS) being the most common subtype (6/10), followed by atrial septal defect with four affected patients and HCM with three affected. Eight out of 16 patients had learning difficulty, of which six were mild (studying in mainstream schools but requiring educational intervention such as additional coaching). Only two patients had moderate to severe learning difficulty (requiring special education). One patient was not assessed as he was too young at the time of enrollment and he did not return for follow-up sessions. The remaining eight had normal intelligence. ## | resequencing of target genes The mean coverage for target bases across the 14 genes was 162x. Heterozygous missense variants were identified in 13 patients: eight were in PTPN11, three in SOS1, and one each for RIT1 and KRAS. The list of identified variants and clinical phenotypes of individual patients are shown in . Seven of the identified variants were de novo, while two were inherited. The inheritance of the identified variants for the remaining four subjects (all PTPN11) was unknown as parental samples were not available. For the two variants that were inherited, the father of patient no. 8 with the PTPN11 M504V (NM_002834.4:c.1510A>G) variant had short stature with typical facial features of NS, while the mother of patient no. 11 with the SOS1 G434R (NM_005633.3:c.1300G>A) variant had short stature, short neck, mild hypertelorism, coarse facial features, and cubitus valgus of NS. One of the patients with a de novo variant in SOS1 also had a paternally inherited NF1 variant (NM_000267.3:c.3484A>G, p.(Met-1162Val)) which is of unknown clinical significance. The father had three café au lait spots >15 mm with right-sided axillary freckling but no other signs of NF1. The child had café au lait spots on his lower back but no freckling. Both the patient and his father did not fulfill the clinical criteria for NF1. ## | genotype-phenotype correlations The number and percentage of patients carrying diseasecausing variants in each of the four mutated genes and their specific phenotypic feature are shown in . The most common phenotypic features are facial dysmorphism, (lowset ears, ocular hypertelorism), short stature, and cardiac defects. The most frequently observed cardiac condition was PS found in five patients. In the eight patients with PTPN11 mutation, two had PS and another two had HCM diagnosed at age 2 and 7 years respectively. Three of the four patients without any identified diseasecausing variant fulfilled the diagnostic criteria for NS. The remaining patient (no. 15 in had lower limb lymphedema and was seen by the geneticist when he was 11 days old. He was negative for other clinical features at that time. As he was lost to follow-up after the second medical review at the age of 3 months, it was uncertain whether additional clinical feature had since developed. # | discussion This study identified disease-causing variants in 13 out of 17 patients with clinical suspicion of NS, similar to other reports where genetic mutations were found in 60%-70% of patients tested. The gene with the largest number of mutations was PTPN11. It is also the main gene that accounts for more than half of identified mutations in other studies [bib_ref] Noonan syndrome-causing genes: Molecular update and an assessment of the, El Bouchikhi [/bib_ref] [bib_ref] Noonan syndrome: Clinical aspects and molecular pathogenesis, Tartaglia [/bib_ref]. For our eight patients with identified PTPN11 disease-causing variants in this study, five of them carried three (Y63C, Q79R, and N308D) of the four T A B L E 1 most common mutations for NS . Seven of the eight disease-causing variants identified in this study were localized in exons 3, 8, and 13, domains identified as hot-spots for de novo mutations [bib_ref] Noonan syndrome-causing genes: Molecular update and an assessment of the, El Bouchikhi [/bib_ref] [bib_ref] Germline KRAS and BRAF mutations in cardio-facio-cutaneous syndrome, Narayanan [/bib_ref]. Three are in the N-amino terminal src-homology 2 domain (N-SH2) encoded by exons 1-3 or the phosphotyrosine phosphatase encoded by exons 8-13. Only the variant found in patient no. 4 is in exon 4 which encodes the C-amino terminal src-homology 2 domain (C-SH2) [bib_ref] Noonan syndrome-causing genes: Molecular update and an assessment of the, El Bouchikhi [/bib_ref]. Three patients had disease-causing variants in SOS1, the second most mutated gene in NS [bib_ref] Noonan syndrome-causing genes: Molecular update and an assessment of the, El Bouchikhi [/bib_ref]. Two of them had the recurrent G434R mutation reported by multiple studies, while the remaining patient had another known mutation [bib_ref] Structure-energy-based predictions and network modelling of RASopathy and cancer missense mutations, Kiel [/bib_ref]. The only disease-causing RIT1 variant found in this study had also been reported [bib_ref] Further evidence of the importance of RIT1 in Noonan syndrome, Bertola [/bib_ref]. One patient in our study had a KRAS mutation which had been previously reported in patients with CFC syndrome . Of the 13 patients with identified disease-causing variants, 10 (76.9%) had short stature, with height of less than third centile based on the growth chart for the local population. No adjustment for parental height could be made as data on parental height was not available. The prevalence of short stature from our study was comparable to that previously reported [bib_ref] Noonan syndrome in diverse populations, Kruszka [/bib_ref] [bib_ref] Noonan syndrome: Clinical features, diagnosis, and management guidelines, Romano [/bib_ref] [bib_ref] Clinical manifestations of mutations in RAS and related intracellular signal transduction factors, Zenker [/bib_ref]. The next most common feature was cardiac abnormalities found in 9 out of 13 patients with identified disease-causing variants. In addition, patient no. 17 for whom no mutation was identified had three cardiac conditions PS, VSD and HCM are all defined in . The high proportion of cardiac abnormalities is not surprising as it is a key feature of NS. This syndrome is also the most common syndromic cause of congenital cardiac conditions after trisomy 21 [bib_ref] Congenital heart diseases in children with Noonan syndrome: An expanded cardiac spectrum..., Marino [/bib_ref]. However, our prevalence of specific cardiac phenotypes stratified by causative genes appeared to be slightly different from other studies. While the 50% proportion for atrial septal defect for patients with PTPN11 mutations was in line with reported findings, only two of eight patients had PS. The prevalence of this clinical feature in PTPN11 mutation carriers had been reported to be 60% [bib_ref] The spectrum of cardiac anomalies in Noonan syndrome as a result of..., Sznajer [/bib_ref] , 63% [bib_ref] New mutations associated with Rasopathies in a Central European population and genotypephenotype..., Cizmarova [/bib_ref] , 65% [bib_ref] Phenotypic spectrum of 80 Greek patients referred as Noonan syndrome and PTPN11..., Papadopoulou [/bib_ref] , 71% [bib_ref] PTPN11 mutations in Noonan syndrome: Molecular spectrum, genotype-phenotype correlation, and phenotypic heterogeneity, Tartaglia [/bib_ref] , and 88% [bib_ref] Genotype-phenotype correlations in Noonan syndrome, Zenker [/bib_ref]. For Korean patients, PS was reported in about 46%-50% of T A B L E 2 Details of phenotypic features in patients with identified mutations (n = 13) patients with PTPN11 mutations [bib_ref] PTPN11, SOS1, KRAS, and RAF1 gene analysis, and genotype-phenotype correlation in Korean..., Ko [/bib_ref] [bib_ref] Spectrum of mutations in Noonan syndrome and their correlation with phenotypes, Lee [/bib_ref]. On the other hand, the same number of our patients (two) had HCM, frequently associated with RAF1 mutations and rare in PTPN11-associated NS [bib_ref] Gain-of-function RAF1 mutations cause Noonan and LEOPARD syndromes with hypertrophic cardiomyopathy, Pandit [/bib_ref] [bib_ref] Noonan syndrome: Clinical aspects and molecular pathogenesis, Tartaglia [/bib_ref]. As the number of patients was small in our study, analysis with larger cohorts of patients would be necessary to establish if the prevalence of PS is indeed lower for NS patients with PTPN11 mutations in Asian populations. Intelligence has been reported to be normal for most patients with NS, with intellectual impairment affecting only about 20% [bib_ref] Noonan syndrome, Allanson [/bib_ref] [bib_ref] Genotype differences in cognitive functioning in Noonan syndrome, Pierpont [/bib_ref]. Seven of our 13 patients with identified disease-causing variants had normal intelligence; the remaining six had some learning difficulty. All three patients with SOS1 disease-causing variants had normal intelligence, consistent with other reports for this gene [bib_ref] Genotype differences in cognitive functioning in Noonan syndrome, Pierpont [/bib_ref] [bib_ref] Gain-of-function SOS1 mutations cause a distinctive form of Noonan syndrome, Tartaglia [/bib_ref]. Four out of eight patients with PTPN11 diseasecausing variants had mild learning difficulty. While PTPN11 N308D is associated with lower IQ in one study [bib_ref] Cognitive profile of disorders associated with dysregulation of the RAS/MAPK signaling cascade, Cesarini [/bib_ref] , one of our two patients with this disease-causing variant had normal intelligence while the other had only mild learning difficulty. The only one with moderate to severe learning difficulty among the 13 patients had a KRAS variant, which is not surprising as it has been reported that learning difficulty is more common in patients with KRAS mutations [bib_ref] Germline KRAS mutations cause Noonan syndrome, Schubbert [/bib_ref]. Moderate to severe cognitive impairment is generally more common in CFC patients and can be used to distinguish CFC from NS . However, Patient no. 13 had typical NS phenotypic features and lacked the skin and hair abnormalities commonly seen in CFC syndrome patients. She also had hydrocephalus requiring ventriculoperitoneal shunt, swallowing dysfunction post fundoplication and gastrostomy, sagittal synostosis postsurgical correction, severe obstructive sleep apnea requiring nocturnal noninvasive continuous positive airway pressure ventilation, and congenital hypoplasia of kidney, which could be present in both NS and CFC syndrome patients. Her case demonstrated the overlapping phenotype of these two syndromes in a patient with KRAS mutation. To our knowledge, this is the first genetic study of NS in a Southeast Asian population. While the spectrum of disease-causing variants in our patients appeared to be similar to populations elsewhere, indicating that there are no population-specific hotspots; the clinical manifestations seemed to be heterogeneous. Our two pairs of patients with identical disease-causing variants did not have similar clinical presentations with each other or with what had been reported in other studies for carriers of this variant. Although the four patients who did not have any mutation identified had fewer clinical features, they might have mutations in genes that were not in the panel. # Acknowledgments This work was supported by NMRC/CG/006/2013 and NMRC/CG/M003/2017 from the Singapore Ministry of Health's National Medical Research Council. We sincerely thank our study subjects and their families for participation and their permission to publish the results. The authors also thank Dr Eddy Saputra Leman (Duke-NUS) for his input and editorial assistance. [fig] T A B L E 1: Demographic and clinical characteristics of all patients (n = 17) at the time of enrollment with their disease-causing variants Reference transcripts: PTPN11: NM_002834.4, SOS1: NM_005633.3, RIT1: NM_006912.5 KRAS: NM_004985.4. ASD: atrial septal defect; HCM: hypertrophic cardiomyopathy; N/A: not applicable; PS: pulmonary stenosis; VSD: ventricular septal defect. [/fig]

Differentiation of Diabetes by Pathophysiology, Natural History, and Prognosis [bib_ref] A 2015 update on predictive molecular pathology and its role in targeted..., Dietel [/bib_ref] [bib_ref] Insulitis and b-cell mass in the natural history of type 1 diabetes, Campbell-Thompson [/bib_ref] ## Pathophysiology of diabetes demographics Type 1 diabetes and type 2 diabetes differentially impact populations based on age, race, ethnicity, geography, and socioeconomic status. ## Type 1 diabetes Between 2001 and 2009, there was a 21% increase in the number of youth with type 1 diabetes in the U.S. [bib_ref] SEARCH for Diabetes in Youth Study. Prevalence of type 1 and type..., Dabelea [/bib_ref]. Its prevalence is increasing at a rate of ;3% per year globally (8). Though diagnosis of type 1 diabetes frequently occurs in childhood, 84% of people living with type 1 diabetes are adults (9). Type 1 diabetes affects males and females equally (10) and decreases life expectancy by an estimated 13 years. An estimated 5-15% of adults diagnosed with type 2 diabetes actually have type 1 diabetes or latent autoimmune diabetes of adults (LADA) (12). Europoid Caucasians have the highest prevalence of type 1 diabetes among U.S. youth, representing 72% of reported cases. Hispanic Caucasians represent 16%, and non-Hispanic blacks represent 9% [bib_ref] SEARCH for Diabetes in Youth Study. Prevalence of type 1 and type..., Dabelea [/bib_ref]. 1-Genetic and environmental risk factors impact inflammation, autoimmunity, and metabolic stress. These states affect b-cell mass and/or function such that insulin levels are eventually unable to respond sufficiently to insulin demands, leading to hyperglycemia levels sufficient to diagnose diabetes. In some cases, genetic and environmental risk factors and gene-environment interactions can directly impact b-cell mass and/or function. Regardless of the pathophysiology of diabetes, chronic high blood glucose levels are associated with microvascular and macrovascular complications that increase morbidity and mortality for people with diabetes. This model positions b-cell destruction and/or dysfunction as the necessary common factor to all forms of diabetes. Incidence and prevalence rates for type 1 diabetes vary dramatically across the globe. At the extremes, China has an incidence of 0.1/100,000 per year and Finland has an incidence of 60/100,000 per year [bib_ref] Incidence of childhood type 1 diabetes worldwide. Diabetes Mondiale (DiaMond) Project Group, Karvonen [/bib_ref]. With some exceptions, type 1 diabetes incidence is positively related to geographic distance north of the equator [bib_ref] Incidence of childhood type 1 diabetes worldwide. Diabetes Mondiale (DiaMond) Project Group, Karvonen [/bib_ref]. Colder seasons are correlated with diagnosis and progression of type 1 diabetes. Both onset of disease and the appearance of islet autoimmunity appear to be higher in autumn and winter than in spring and summer [bib_ref] Age-adjusted rates of diagnosed diabetes per 100 civilian, non-institutionalized population, by sex, Knip [/bib_ref]. ## Type 2 diabetes In the U.S., an estimated 95% of the nearly 30 million people living with diabetes have type 2 diabetes. An additional 86 million have prediabetes, putting them at high risk for developing type 2 diabetes (9). Among the demographic associations for type 2 diabetes are older age, race/ ethnicity, male sex, and socioeconomic status (9). Type 2 diabetes incidence is increasing in youth, especially among the racial and ethnic groups with disproportionately high risk for developing type 2 diabetes and its complications: American Indians, African Americans, Hispanics/Latinos, Asians, and Pacific Islanders (9). Older age is very closely correlated to risk for developing type 2 diabetes. More than one in four Americans over the age of 65 years have diabetes, and more than half in this agegroup have prediabetes (9). The prevalence of type 2 diabetes in the U.S. is higher for males (6.9%) than for females (5.9%) (15). There is a high degree of variability for prevalence of type 2 diabetes across the globe. East Asia, South Asia, and Australia have more adults with diabetes than any other region (153 million). North America and the Caribbean have the highest prevalence rate, with one in eight affected (8). Independent of geography, the risk of developing type 2 diabetes is associated with low socioeconomic status. Low educational level increases risk by 41%, low occupation level by 31%, and low income level by 40% [bib_ref] Type 2 diabetes incidence and socio-economic position: a systematic review and meta-analysis, Agardh [/bib_ref]. ## Research gaps The assembled experts agreed that research efforts are needed to define causative factors that account for the established correlations among different demographic subsets and the corresponding variable risks for diabetes. Factors associated with race/ethnicity and geography that differentially increase risk for type 1 diabetes and for type 2 diabetes need to be defined. For type 2 diabetes, the drivers of increased risk in individuals of low socioeconomic status also need to be established. ## Genetics Both type 1 and type 2 diabetes are polygenic diseases where many common variants, largely with small effect size, contribute to overall disease risk. Disease heritability (h 2 ), defined as sibling-relative risk, is 3 for type 2 diabetes and 15 for type 1 diabetes [bib_ref] Genetics of type 2 diabetes-pitfalls and possibilities, Prasad [/bib_ref]. The lifetime risk of developing type 2 diabetes is ;40% if one parent has type 2 diabetes and higher if the mother has the disease [bib_ref] Metabolic consequences of a family history of NIDDM (the Botnia study): evidence..., Groop [/bib_ref]. The risk for type 1 diabetes is ;5% if a parent has type 1 diabetes and higher if the father has the disease [bib_ref] Autoimmunity and familial risk of type 1 diabetes, Hämäläinen [/bib_ref]. Maturity-onset diabetes of the young (MODY) is a monogenic disease and has a high h 2 of ;50 [bib_ref] Familial risks for type 2 diabetes in Sweden, Hemminki [/bib_ref]. Mutations in any 1 of 13 different individual genes have been identified to cause MODY [bib_ref] Clinical implications of a molecular genetic classification of monogenic beta-cell diabetes, Murphy [/bib_ref] , and a genetic diagnosis can be critical for selecting the most appropriate therapy. For example, children with mutations in KCJN11 causing MODY should be treated with sulfonylureas rather than insulin. ## Type 1 diabetes The higher type 1 diabetes prevalence observed in relatives implies a genetic risk, and the degree of genetic identity with the proband correlates with risk [bib_ref] Diabetes in identical twins. A study of 200 pairs, Barnett [/bib_ref] [bib_ref] Islet-cell antibodies and beta-cell function in monozygotic triplets and twins initially discordant..., Srikanta [/bib_ref] [bib_ref] 25. Rich SS. Mapping genes in diabetes. Genetic epidemiological perspective, Redondo [/bib_ref] [bib_ref] Extreme genetic risk for type 1A diabetes, Aly [/bib_ref]. Gene variants in one major locus, human leukocyte antigen (HLA) [bib_ref] Type 1 Diabetes Genetics Consortium. HLA class I and genetic susceptibility to..., Noble [/bib_ref] , confer 50-60% of the genetic risk by affecting HLA protein binding to antigenic peptides and antigen presentation to T cells [bib_ref] Additive and interaction effects at three amino acid positions in HLA-DQ and..., Hu [/bib_ref]. Approximately 50 additional genes individually contribute smaller effects (25,29). These contributors include gene variants that modulate immune regulation and tolerance [bib_ref] Defects in IL-2R signaling contribute to diminished maintenance of FOXP3 expression in..., Long [/bib_ref] [bib_ref] An autoimmune-associated variant in PTPN2 reveals an impairment of IL-2R signaling in..., Long [/bib_ref] [bib_ref] The insulin gene is transcribed in the human thymus and transcription levels..., Pugliese [/bib_ref] [bib_ref] Type 1 diabetes: primary antigen/peptide/ register/trimolecular complex, Sosinowski [/bib_ref] , variants that modify viral responses [bib_ref] MDA5 and PTPN2, two candidate genes for type 1 diabetes, modify pancreatic..., Colli [/bib_ref] [bib_ref] TYK2, a candidate gene for type 1 diabetes, modulates apoptosis and the..., Marroqui [/bib_ref] , and variants that influence responses to environmental signals and endocrine function [bib_ref] Genes affecting b-cell function in type 1 diabetes, Fløyel [/bib_ref] , as well as some that are expressed in pancreatic b-cells [bib_ref] Candidate genes for type 1 diabetes modulate pancreatic islet inflammation and b-cell..., Santin [/bib_ref]. Genetic influences on the triggering of islet autoimmunity and disease progression are being defined in relatives [bib_ref] Role of type 1 diabetes-associated SNPs on risk of autoantibody positivity in..., Törn [/bib_ref] [bib_ref] Effects of non-HLA gene polymorphisms on development of islet autoimmunity and type..., Steck [/bib_ref]. Together, these gene variants explain ;80% of type 1 diabetes heritability. Epigenetic [bib_ref] Genetic and epigenetic factors in etiology of diabetes mellitus type 1, Stankov [/bib_ref] , gene expression, and regulatory RNA profiles (36) may vary over time and reflect disease activity, providing a dynamic readout of risk. Genetic variants can also identify patients at higher risk, predict rates of C-peptide decline, and predict response to various therapies [bib_ref] Staging presymptomatic type 1 diabetes: a scientific statement of JDRF, the, Insel [/bib_ref]. With a better understanding of inheritance profiles, it may become possible to realize new targets for individualized intervention. ## Type 2 diabetes While a subset of genetic variants are linked to both type 1 and type 2 diabetes [bib_ref] Association of TCF7L2 variation with single islet autoantibody expression in children with..., Redondo [/bib_ref] [bib_ref] Overlap of genetic susceptibility to type 1 diabetes, type 2 diabetes, and..., Basile [/bib_ref] , the two diseases have a largely distinct genetic basis, which could be leveraged toward classification of diabetes [bib_ref] A type 1 diabetes genetic risk score can aid discrimination between type..., Oram [/bib_ref]. Genome-wide association studies have identified more than 130 genetic variants associated with type 2 diabetes, glucose levels, or insulin levels; however, these variants explain less than 15% of disease heritability [bib_ref] DIAbetes Genetics Replication and Meta-analysis (DIAGRAM) Consortium. Large-scale association analyses identify new..., Scott [/bib_ref] [bib_ref] DIAbetes Genetics Replication And Meta-analysis (DIAGRAM) Consortium. Genetic fine mapping and genomic..., Gaulton [/bib_ref]. There are many possibilities for explaining the majority of type 2 diabetes heritability, including disease heterogeneity, gene-gene interactions, and epigenetics. Most type 2 variants are in noncoding genomic regions. Some variants, such as those in KCNQ1, show strong parent-of-origin effects [bib_ref] Insights into the molecular mechanism for type 2 diabetes susceptibility at the..., Travers [/bib_ref]. It is possible that children of mothers carrying KCNQ1 are born with a reduced functional b-cell mass and thereby are less able to increase their insulin secretion when exposed to insulin resistance [bib_ref] Early-life origins of type 2 diabetes: fetal programming of the beta-cell mass, Portha [/bib_ref]. Another area of particular interest has been the search for rare variants protecting from type 2 diabetes, such as loss-of-function mutations in SLC30A8 [bib_ref] Go-T2D Consortium; T2D-GENES Consortium. Loss-of-function mutations in SLC30A8 protect against type 2..., Flannick [/bib_ref] , which could offer potential new drug targets for type 2 diabetes. To date, however, the improvement in predictive value of known genetic variants over that of classic clinical risk factors (BMI, family history, glucose) has proven minimal in type 2 diabetes. The rapid development of molecular genetic tools and decreasing costs for next-generation sequencing should make dissection of the black box of genetics of diabetes possible in the near future, but at this point, apart from the profiles that distinguish between type 1 and type 2 diabetes and a limited number of specific variants that identify small subgroups of patients (MODY), genetics has not been successful in further differentiating subclasses of diabetes. ## Research gaps After consideration of the known genetic associations with diabetes risk, consensus developed that the field is not yet at a place where genetics has provided actionable information to guide treatment decisions, with a few notable exceptions, namely in MODY. The experts agreed there is a need to use the increasingly accessible and affordable technologies to further refine our understanding of how genetic variations affect the rate of progression of diabetes and its complications. The expert committee also highlighted the importance of determining categorical phenotypic subtypes of diabetes in order to link specific genetic associations to these phenotypic subtypes. These types of information are necessary to develop the tools to predict response to-and side effects of-therapeutic approaches for diabetes in patient populations. ## Environmental influences Despite the genetic underpinnings of the diseases, the prevalence of both type 1 and type 2 diabetes is increasing globally at a rate that outpaces genetic variation, suggesting that environmental factors also play a key role in both types of diabetes. Common environmental factors are associated with type 1 and type 2 diabetes, including dietary factors, endocrine disruptors and other environmental polluters, and gut microbiome composition. In addition to well-established roles in type 2 diabetes, obesity and insulin resistance may be accelerators of type 1 diabetes. Conversely, islet autoimmunity associated with possible environmental triggers (e.g., diet, infection) may have a role in a subset of people diagnosed with type 2 diabetes. ## Type 1 diabetes Discordance rates in twins, the rise in global incidence, variance in geographic prevalence, and assimilation of local disease incidence rates when individuals migrate from low-to high-incidence countries all support an environmental influence on risk for developing type 1 diabetes. Furthermore, many lines of evidence suggest that environmental factors interact with genetic factors in both the triggering of autoimmunity and the subsequent progression to type 1 diabetes. Supporting this gene-environment interaction is the fact that most subjects with the highest-risk HLA haplotypes do not develop type 1 diabetes. The timing of exposure to environmental triggers may also be critical. The variability of age at disease onset complicates the study of environmental exposures, though the early age of onset of islet autoantibodies associated with childhood-onset type 1 diabetes suggests that environmental exposures in the first few years of life may be contributors. Among the environmental associations linked to type 1 diabetes are enteroviral and other infections [bib_ref] Potential viral pathogenic mechanism in human type 1 diabetes, Schneider [/bib_ref] [bib_ref] Virus infections and type 1 diabetes risk, Roivainen [/bib_ref] and altered intestinal microbiome composition [bib_ref] Toward defining the autoimmune microbiome for type 1 diabetes, Giongo [/bib_ref]. The timing of exposure to foods including cereal (54) and nutrients such as gluten (55) may influence b-cell autoimmunity. Low serum concentrations of vitamin D have been linked to type 1 diabetes. Perinatal risk factors and toxic doses of nitrosamine compounds have been implicated in the genesis of diabetes. The effects of any environmental toxin on type 1 diabetes need further exploration. Studies on the environmental contributions to type 1 diabetes have been small and somewhat contradictory, highlighting the need for larger collaborative investigations such as The Environmental Determinants of Diabetes in the Young (TEDDY) [bib_ref] TEDDY-The Environmental Determinants of Diabetes in the Young: an observational clinical trial, Hagopian [/bib_ref] , which aims to identify infectious agents, dietary factors, and other environmental factors that trigger islet autoimmunity and/or type 1 diabetes. ## Type 2 diabetes Type 2 diabetes develops when b-cells fail to secrete sufficient insulin to keep up with demand, usually in the context of increased insulin resistance. A minority of people diagnosed with type 2 diabetes also have evidence of islet autoimmunity [bib_ref] Identification of tyrosine phosphatase 2 (256-760) construct as a new, sensitive marker..., Tiberti [/bib_ref]. Obesity is a major risk factor for type 2 diabetes (59,60) with complex genetic and environmental etiology. Insulin resistance develops with ectopic fat deposition in the liver and muscle. Fat may also accumulate in the pancreas and contribute to the decline in b-cell function, islet inflammation, and eventual b-cell death (61). Type 2 diabetes occurs at different levels of BMI/body fat composition in different individuals and at lower BMI for Asians and Asian Americans (62). For susceptible people, there may be a personal "fat threshold" at which ectopic fat accumulation occurs, worsening insulin resistance and resulting in b-cell decompensation. Weight loss improves insulin sensitivity in liver and skeletal muscle [bib_ref] Effects of weight loss on mechanisms of hyperglycemia in obese non-insulin-dependent diabetes..., Henry [/bib_ref] and may also reduce pancreatic fat accumulation [bib_ref] Diabetes Prevention Program Research Group. TCF7L2 polymorphism, weight loss and proinsulin: insulin..., Lim [/bib_ref]. Defects in insulin secretion are at least partially reversible with energy restriction and weight loss in prediabetes and recent-onset type 2 diabetes (65). Unfortunately, it is difficult to reverse long-standing diabetes, even with the large weight loss associated with bariatric surgery (66). Both reduced sleep time and increased sleep time are associated with the development of obesity and diabetes. Obstructive sleep apnea reduces sleep time and sleep quality and is associated with type 2 diabetes and metabolic syndrome. The modern "24-hour culture" may reduce sleep time and thereby also contribute to increased risk of type 2 diabetes. And while associations with additional environmental factors exist, there have been no direct causal relationships defined to date. ## Research gaps There is a clear correlation of environmental influences to diabetes risk. Yet, the assembled experts agreed that hypothesis-driven research is needed to define direct causal relationships between specific environmental factors and pathophysiologies leading to diabetes. Research efforts need to address environmental etiologies of type 1 diabetes and determine their relative contribution to onset of autoimmunity and progression to symptomatic disease. Whether there is a direct causal role of the intestinal microbiota in pathogenesis of type 1 and type 2 diabetes and response to therapies needs to be determined. Public health interventions that successfully reduce the levels of consumption of energy-dense foods and/or reduce sedentary time and increase time spent in physical activity need to be evaluated to determine whether they can reduce type 2 diabetes incidence at a population level. ## Natural history and prognosis Regardless of the particular pathophysiology of an individual's diabetes, the unifying characteristic of the vast majority of diabetes is hyperglycemia resulting from b-cell destruction or dysfunction. There is a continuum of progressive dysglycemia as insulin insufficiency increases over time. Understanding the natural history related to b-cell mass and function is key to staging the diseases and identifying where and how interventions can best be made to prevent or delay disease progression and complications. ## B-cell mass and function While type 1 diabetes results from immune-mediated destruction of b-cells and type 2 diabetes is primarily associated with glucose-specific insulin secretory defects, there is growing evidence of significant overlap across the spectrum of diabetes. For example, b-cell mass is also reduced in people with type 2 diabetes (67). In both type 1 and type 2 diabetes, the stress response induced by hyperglycemia may play a role in b-cell apoptosis [bib_ref] Increased expression of antioxidant and antiapoptotic genes in islets that may contribute..., Laybutt [/bib_ref]. Changes in b-cell phenotype associated with hyperglycemia may reflect a dedifferentiation of b-cells important to the natural history and staging of diabetes [bib_ref] Five stages of evolving b-cell dysfunction during progression to diabetes, Weir [/bib_ref]. Clearly, an insufficient number or functional decline of b-cells is central to hyperglycemia and the downstream complications of diabetes. Understanding the state of the b-cell is key to defining subtypes of diabetes. ## Type 1 diabetes Abnormal insulin secretion can occur well before the diagnosis of type 1 diabetes [bib_ref] Predictive value of intravenous glucose tolerance test insulin secretion less than or..., Vardi [/bib_ref] [bib_ref] Diagnosis of pre-type I diabetes, Chase [/bib_ref] [bib_ref] Firstdegree relatives of patients with type I diabetes mellitus. Islet-cell antibodies and..., Srikanta [/bib_ref] [bib_ref] Triad of markers for identifying children at high risk of developing insulin-dependent..., Ginsberg-Fellner [/bib_ref] , with a gradual decline beginning at least 2 years before diagnosis and accelerating proximal to diagnosis [bib_ref] Type 1 Diabetes TrialNet and Diabetes Prevention Trial-Type 1 Study Groups. Acceleration..., Sosenko [/bib_ref]. A decline in b-cell sensitivity to glucose (76) appears to occur on a similar timeframe. As the early insulin response falters, the later insulin response becomes greater, indicating a possible compensatory mechanism. The accelerated loss of insulin response continues into the early postdiagnostic period [bib_ref] Glucose and C-peptide changes in the perionset period of type 1 diabetes..., Sosenko [/bib_ref]. Insulin secretion decline during the first few years after diagnosis has been described as biphasic, steeper during the first year than during the second year after diagnosis. Data also suggest that the rate of decline is slower in adults [bib_ref] Type 1 Diabetes TrialNet Study Group. Fall in C-peptide during first 2..., Greenbaum [/bib_ref]. The loss of insulin secretion can continue for years after diagnosis until little or no insulin secretion remains. However, low levels of C-peptide are detectable in the majority of patients after 30 years of type 1 diabetes [bib_ref] The majority of patients with longduration type 1 diabetes are insulin microsecretors..., Oram [/bib_ref]. Glucose levels are also frequently elevated years before the diagnosis of type 1 diabetes (80-82). Even within the normal range, higher glucose levels are predictive of type 1 diabetes [bib_ref] Diabetes Prevention Trial-Type 1 Study Group. Increasing the accuracy of oral glucose..., Sosenko [/bib_ref]. There are wide fluctuations of glucose during the progression to type 1 diabetes [bib_ref] Diabetes Prevention Trial-Type 1 Study Group. Glucose excursions between states of glycemia..., Sosenko [/bib_ref]. Metabolic markers of progression, such as the occurrence of dysglycemia, could be utilized to more precisely predict the onset of diabetes in at-risk individuals [bib_ref] Staging presymptomatic type 1 diabetes: a scientific statement of JDRF, the, Insel [/bib_ref] [bib_ref] Diabetes Prevention Trial-Type 1 Study Group. Incident dysglycemia and progression to type..., Sosenko [/bib_ref]. Risk scores that combine dynamic changes in glucose and C-peptide can further enhance prediction [bib_ref] Type 1 Diabetes TrialNet and Diabetes Prevention Trial-Type 1 Study Groups. Validation..., Sosenko [/bib_ref]. ## Type 2 diabetes Defective insulin secretion is central to the pathophysiology of type 2 diabetes. To maintain normal glucose levels, insulin secretion varies over a wide range in response to insulin sensitivity. The relationship between insulin secretion and insulin sensitivity is curvilinear and is expressed as the disposition index. People with type 2 diabetes cannot adequately increase insulin secretion to overcome insulin resistance and have a low disposition index [bib_ref] Effects of type 2 diabetes on insulin secretion, insulin action, glucose effectiveness,..., Basu [/bib_ref]. Consequently, while absolute insulin levels may be higher in obese subjects with type 2 diabetes who are insulin resistant than they are in lean control subjects who are insulin sensitive, they are lower than appropriate for their degree of insulin resistance. Firstphase insulin secretion, especially in response to stimulation by glucose, is markedly impaired or lost (89). Maximal insulin secretion and potentiation by hyperglycemia of insulin responses to nonglucose stimuli are severely reduced [bib_ref] Diminished B cell secretory capacity in patients with noninsulin-dependent diabetes mellitus, Ward [/bib_ref] , and the ratio of proinsulin to insulin (C-peptide) is high in type 2 diabetes [bib_ref] Serum proinsulin levels at fasting and after oral glucose load in patients..., Yoshioka [/bib_ref]. Over time, hyperglycemia tends to become more severe and more difficult to treat. This progressive nature of type 2 diabetes is usually due to ongoing deterioration of b-cell function. While prediabetes and diabetes are diagnosed by absolute thresholds (92), dysglycemia is a continuum progressing from normal to overt diabetes. Early screening offers a window for treatment that may prevent or delay progression of the disease and its complications [bib_ref] We can change the natural history of type 2 diabetes, Phillips [/bib_ref] [bib_ref] Are current diagnostic guidelines delaying early detection of dysglycemic states? Time for..., Bergman [/bib_ref]. In prediabetes, impaired glucose tolerance or impaired fasting glucose indicates glucose levels higher than normal but not in the diabetes range. Currently, most clinicians do not treat these patients to completely control blood glucose levels. Even after initiation of therapy in frank diabetes, intensification of therapy is often delayed [bib_ref] It's time to overcome clinical inertia, Phillips [/bib_ref] [bib_ref] Delay of insulin addition to oral combination therapy despite inadequate glycemic control:..., Nichols [/bib_ref] [bib_ref] Clinical inertia in people with type 2 diabetes: a retrospective cohort study..., Khunti [/bib_ref] , exposing people to hyperglycemia for years [bib_ref] We can change the natural history of type 2 diabetes, Phillips [/bib_ref]. Several studies have shown that treatment with lifestyle change or medication can reduce the progression from prediabetes to diabetes [bib_ref] Finnish Diabetes Prevention Study Group. Prevention of type 2 diabetes mellitus by..., Tuomilehto [/bib_ref] [bib_ref] Diabetes Prevention Program Research Group. Reduction in the incidence of type 2..., Knowler [/bib_ref]. Furthermore, a clinical benefit of early therapy has been demonstrated (100,101), with reductions in retinopathy and cardiovascular and allcause mortality [bib_ref] Cardiovascular mortality, all-cause mortality, and diabetes incidence after lifestyle intervention for people..., Li [/bib_ref]. This evidence suggests that identifying prediabetes at an early stage and keeping glucose levels close to normal could change the natural history of the disease (93). ## Research gaps. The strong consensus of this group was that the primary defect resulting in hyperglycemia is insufficient b-cell number and/or b-cell function (of various etiologies). From this b-cell-centric view, it is imperative to determine what etiological factors are the basis for abnormal insulin secretion patterns in type 1 diabetes and type 2 diabetes. Biomarkers and imaging tools are needed to assess b-cell mass and loss of functional mass and to monitor progression and response to therapeutic interventions. The point at which b-cell dysfunction becomes irreversible needs to be determined. The molecular basis for the glucose-specific insulin secretory defect and the role of b-cell dedifferentiation in type 1 diabetes and in type 2 diabetes need to be determined. The extent to which insulin resistance contributes to glycemia and the complications of type 1 diabetes remains unknown. Research is needed to determine whether increased b-cell activity, stimulated by insulin resistance, enhances or accelerates the b-cell lesion in type 1 diabetes and in type 2 diabetes and to identify mechanisms by which b-cells can overcome an insulin-resistant environment. ## Autoimmunity Circulating autoantibodies against insulin, glutamic acid decarboxylase (GAD), the protein tyrosine phosphatase IA-2, and/or zinc transporter 8 can be detected prior to clinical diagnosis of type 1 diabetes [bib_ref] Predicting type 1 diabetes using biomarkers, Bonifacio [/bib_ref]. While individuals with single autoantibody positivity frequently revert to negative, reversion is rare in people with multiple autoantibodies [bib_ref] TEDDY Study Group. Reversion of b-cell autoimmunity changes risk of type 1..., Vehik [/bib_ref]. Currently, we lack sufficient biomarkers and imaging techniques to monitor autoantibody flare-ups, reversions, and progression to type 1 diabetes. The presence of two or more islet autoantibodies in children with HLA risk genotypes or with relatives who have type 1 diabetes is associated with a 75% risk of developing clinical diabetes within 10 years [bib_ref] Seroconversion to multiple islet autoantibodies and risk of progression to diabetes in..., Ziegler [/bib_ref]. Risk is incremental with detection of increasing numbers of autoantibodies [bib_ref] Seroconversion to multiple islet autoantibodies and risk of progression to diabetes in..., Ziegler [/bib_ref] [bib_ref] TEDDY Study Group. Predictors of progression from the appearance of islet autoantibodies..., Steck [/bib_ref]. A positive test for at least two autoantibodies is now considered a diagnostic stage of type 1 diabetes [fig_ref] Table 1 -: Staging of type 1 diabetes Fasting plasma glucose 100-125 mg/dL c 2-h... [/fig_ref] [bib_ref] Staging presymptomatic type 1 diabetes: a scientific statement of JDRF, the, Insel [/bib_ref]. The presence of islet autoantibodies reflects an underlying immune B-and T-cell response to b-cell antigens. Autoimmune responses to b-cells lead to loss of b-cell mass and function and onset of glucose intolerance, representing the next distinct stage prior to onset of clinical symptoms of diabetes. Despite the strong prognostic value of autoimmunity in type 1 diabetes, there is no successful strategy to prevent or treat it. HLA confers strong susceptibility for the development of two or more islet autoantibodies [bib_ref] Islet autoimmunity in infants with a type I diabetic relative is common..., Colman [/bib_ref]. For primary prevention of b-cell autoimmunity in children, data suggest there may be a critical period in the first 2 years of life [bib_ref] Age-related islet autoantibody incidence in offspring of patients with type 1 diabetes, Ziegler [/bib_ref] [bib_ref] Early seroconversion and rapidly increasing autoantibody concentrations predict prepubertal manifestation of type..., Parikka [/bib_ref] [bib_ref] TEDDY Study Group. The 6 year incidence of diabetes-associated autoantibodies in genetically..., Krischer [/bib_ref]. Interestingly, autoantibodies against GAD are present in ;5% of individuals diagnosed with type 2 diabetes [bib_ref] Diabetes at the crossroads: relevance of disease classification to pathophysiology and treatment, Leslie [/bib_ref]. As compared with GAD antibody-negative patients with type 2 diabetes, these patients have lower BMI and residual b-cell function. Further, they carry a genetic profile more similar to that of patients with type 1 diabetes and an earlier requirement for insulin therapy [bib_ref] Diabetes at the crossroads: relevance of disease classification to pathophysiology and treatment, Leslie [/bib_ref] , suggesting that autoimmune diabetes in adults may actually be a form of type 1 diabetes that exhibits slow progression associated with later age of onset. ## Research gaps The assembled group agreed that while it is clear that inflammation and autoimmunity lead to b-cell destruction c Clinical symptoms c Diabetes by standard criteria characteristic of type 1 diabetes, much more information is needed to understand the pathophysiology and progression of autoimmunity related to diabetes in order to develop rational approaches to prevent or reverse it. We do not have a clear understanding of whether different antigenic targets, single-antibody positivity, or other contributing factors have variable prognostic, genetic and environmental correlates that can be used to better develop and apply stage-appropriate personalized therapies. The molecular mechanisms by which b-cells die or fail in the presence of b-cell autoimmunity need determination. Biomarkers and imaging tools are needed to define reversion or stable autoimmunity versus active or flaring autoimmunity. Furthermore, inexpensive specific and sensitive assays to identify b-cell autoimmunity are needed, to be deployed on a population-wide level and beyond the confines of specialized laboratories. ## Therapeutics Aside from insulin and insulin analogs, therapies for diabetes include those that enhance insulin secretion, those that stimulate insulin action, those that reduce hepatic and endogenous glucose production, and those that impact glycemia through other mechanisms. By better understanding the pathophysiology and natural history of various subtypes of diabetes and applying what we know about the modes of action and pharmacogenomics of existing therapies, we can better apply a personalized approach to diabetes management. There is a growing body of evidence regarding which phenotypic and genotypic subsets of patients with diabetes respond best, or are resistant to, specific therapies [bib_ref] Personalized medicine in diabetes: the role of 'omics' and biomarkers, Pearson [/bib_ref] , including sulfonylureas [bib_ref] No deterioration in glycemic control in HNF-1a maturity-onset diabetes of the young..., Shepherd [/bib_ref] [bib_ref] Impact of clinical factors and CYP2C9 variants for the risk of severe..., Holstein [/bib_ref] , metformin [bib_ref] Association of organic cation transporter 1 with intolerance to metformin in type..., Dujic [/bib_ref] [bib_ref] Recessive mutations in the cancer gene Ataxia Telangiectasia Mutated (ATM), at a..., Connelly [/bib_ref] , thiazolidinediones [bib_ref] Addition of rosiglitazone to metformin is most effective in obese, insulin-resistant patients..., Jones [/bib_ref] [bib_ref] Attenuation of islet-specific T cell responses is associated with C-peptide improvement in..., Brooks-Worrell [/bib_ref] , incretin therapies [bib_ref] PRIBA Study Group. Markers of b-cell failure predict poor glycemic response to..., Jones [/bib_ref] , and inhibitors of sodium-glucose cotransporter 2 (SGLT2) [bib_ref] Effectiveness of ipragliflozin, a sodium-glucose co-transporter 2 inhibitor, as a second-line treatment..., Ohki [/bib_ref] [bib_ref] Dapagliflozin 006 Study Group. Longterm efficacy of dapagliflozin in patients with type..., Wilding [/bib_ref]. ## Type 1 diabetes Individuals with type 1 diabetes require intensive therapy, characterized by exogenous insulin administration through multiple daily injections with both fast-acting insulin with meals and basal insulin, or with continuous subcutaneous insulin infusion through pumps. There are no significant generalizable differences in efficacy or safety between the two approaches [bib_ref] Comparative effectiveness and safety of methods of insulin delivery and glucose monitoring..., Yeh [/bib_ref]. The goal of intensive insulin therapy is to maintain as close to normal glucose concentration as possible while avoiding hypoglycemia. Achieving this goal requires individualization of treatment and targets, which may also change over time within individuals. The American Diabetes Association's glycemic target for adults is HbA 1c ,7%. However, consideration of individual circumstances is critical. Pediatric patients are recommended to target ,7.5%, whereas adults who are able to do so safely should target ,6.5%. Both long-acting and short-acting insulin analog preparations with more predictable time-action profiles have been developed, allowing patients to achieve more physiological insulin delivery and, therefore, tighter glucose control with fewer side effects. Technologies for self-monitoring blood glucose and continuous glucose monitoring have advanced in recent years and are becoming more widespread. Continuous glucose monitoring allows patients to visualize changes in glucose levels and tailor their treatment in real time [bib_ref] Juvenile Diabetes Research Foundation Continuous Glucose Monitoring Study Group. Continuous glucose monitoring..., Tamborlane [/bib_ref]. The amylin analog pramlintide is approved for use as an adjunct to insulin in patients with type 1 diabetes who have not achieved glycemic goals despite optimized insulin therapy. Pramlintide lowers postprandial glucose [bib_ref] Pramlintide reduces postprandial glucose excursions when added to regular insulin or insulin..., Weyer [/bib_ref] , thereby improving overall glycemic control, and it has a modest but significant weight loss effect. However, pramlintide added to insulin may increase the risk of hypoglycemia [bib_ref] Amylin replacement with pramlintide as an adjunct to insulin therapy improves long-term..., Ratner [/bib_ref] [bib_ref] A double-blind, placebo-controlled trial assessing pramlintide treatment in the setting of intensive..., Edelman [/bib_ref]. A number of agents currently approved for the treatment of type 2 diabetes have also been investigated for use in type 1 diabetes, including a-glucosidase inhibitors [bib_ref] Nocturnal hypoglycemia in type 1 diabetes: an assessment of preventive bedtime treatments, Raju [/bib_ref] [bib_ref] Efficacy and safety of acarbose in the treatment of Type 1 diabetes..., Riccardi [/bib_ref] , thiazolidinediones [bib_ref] The effect of rosiglitazone on overweight subjects with type 1 diabetes, Strowig [/bib_ref] [bib_ref] Pioglitazone may accelerate disease course of slowly progressive type 1 diabetes, Shimada [/bib_ref] [bib_ref] Rosiglitazone preserves islet betacell function of adult-onset latent autoimmune diabetes in 3..., Yang [/bib_ref] , metformin [bib_ref] The use of metformin in type 1 diabetes: a systematic review of..., Vella [/bib_ref] , glucagon-like peptide 1 (GLP-1) receptor agonists [bib_ref] Four weeks of treatment with liraglutide reduces insulin dose without loss of..., Kielgast [/bib_ref] [bib_ref] GLP-1 receptor agonists in type 1 diabetes: a proof-of-concept approach, Crisci [/bib_ref] , dipeptidyl peptidase 4 (DPP-4) inhibitors [bib_ref] Effect of sitagliptin on post-prandial glucagon and GLP-1 levels in patients with..., Garg [/bib_ref] , and SGLT2 inhibitors [bib_ref] Remogliflozin etabonate, a selective inhibitor of the sodium-glucose transporter 2, improves serum..., Mudaliar [/bib_ref] [bib_ref] Diabetic ketoacidosis with canagliflozin, a sodium-glucose cotransporter 2 inhibitor, in patients with..., Peters [/bib_ref]. The benefits of these agents in type 1 diabetes are not well established, and their eventual use in this population will depend on further demonstration of efficacy and safety. ## Type 2 diabetes There are many agents now available to treat hyperglycemia in type 2 diabetes, with varying mechanisms of action and targeting different pathophysiological components of the disease. Many agents are not always able to achieve adequate control unless they are started earlier in disease progression or are used in combinations (metformin, SGLT2 inhibitors, DPP-4 inhibitors, GLP-1 receptor agonists, peroxisome proliferator-activated receptor g agonists). This limitation in efficacy may be due in part to the fact that these agents are often initiated after b-cell function or mass has deteriorated beyond a critical level or to their limited effects on insulin secretion. Many people with type 2 diabetes ultimately require insulin therapy, which reflects long-standing type 2 diabetes and greatly diminished b-cell function but also likely includes individuals who have slowly progressing autoimmune diabetes with adult onset (LADA) or other ambiguous forms of diabetes. Age. Data from randomized controlled trials in people with type 2 diabetes under the age of 18 years or over the age of 65 years are scarce. Beneficial effects of tight glucose control on complications take years to be realized [bib_ref] 10-year follow-up of intensive glucose control in type 2 diabetes, Holman [/bib_ref]. Targets of glucose control should be adapted to life expectancy, frailty, biological age, and social situation rather than just calendar age. HbA 1c targets in this population need to be adjusted when using agents that cause side effects such as hypoglycemia. However, overt hyperglycemia needs to be addressed to avoid acute complications of diabetes and a catabolic state [bib_ref] Impact of glucose level on morbidity and mortality in elderly with diabetes..., Twito [/bib_ref]. Comorbidities: Kidney Impairment. Kidney impairment is a prevalent complication of diabetes. It is also an independent comorbidity, very often caused by vascular complications in people with type 2 diabetes. Therapeutic choices become more limited because of contraindications (e.g., metformin) or the need for good kidney function for efficacy (e.g., SGLT2 inhibitors), leaving many patients with only insulin therapy [bib_ref] Noninsulin glucose-lowering agents for the treatment of patients on dialysis, Flynn [/bib_ref]. Targets for glucose control in the population with kidney impairment may need to be adapted, as kidney impairment also predisposes to hypoglycemia. The use of HbA 1c is also problematic in people with kidney impairment because of reduced red blood cell survival, use of erythropoietin, modifications of hemoglobin (e.g., carbamylation), and mechanical destruction of red blood cells on dialysis [bib_ref] Influence of in vivo hemoglobin carbamylation on HbA1c measurements by various methods, Chachou [/bib_ref]. Comorbidities: Cardiovascular Complications. Cardiovascular complications require a multifactorial approach, including blood pressure and lipid control. Hypoglycemia is linked to arrhythmias and mortality in people with a history of cardiovascular events [bib_ref] Risk of cardiac arrhythmias during hypoglycemia in patients with type 2 diabetes..., Chow [/bib_ref]. However, when agents that do not cause hypoglycemia can be used, tight glucose control should be sought. Agents such as DPP-4 inhibitors [bib_ref] SAVOR-TIMI 53 Steering Committee and Investigators. Saxagliptin and cardiovascular outcomes in patients..., Scirica [/bib_ref] [bib_ref] Alogliptin after acute coronary syndrome in patients with type 2 diabetes, White [/bib_ref] [bib_ref] Effect of sitagliptin on cardiovascular outcomes in type 2 diabetes, Green [/bib_ref] and GLP-1 receptor agonists [bib_ref] Lixisenatide in patients with type 2 diabetes and acute coronary syndrome, Pfeffer [/bib_ref] have been shown to be safe in this population. Some agents, such as pioglitazone [bib_ref] PROactive Investigators. Secondary prevention of macrovascular events in patients with type 2..., Dormandy [/bib_ref] and metformin, may even be cardioprotective. Empagliflozin [bib_ref] EMPA-REG OUTCOME Investigators. Empagliflozin, cardiovascular outcomes, and mortality in type 2 diabetes, Zinman [/bib_ref] and liraglutide (153) reduce cardiovascular and all-cause mortality over 2.5-5 years of therapy in patients at high risk of cardiovascular disease. Nephropathy is a recognized risk factor for cardiovascular complications, especially in type 1 diabetes. Weight. To avoid comorbidities and complications associated with obesity, weight management should be a priority in all patients, independent of BMI. Weight loss can be achieved by lifestyle intervention, choosing glucoselowering drugs that promote weight loss, and incorporating obesity pharmacotherapy or bariatric surgery in appropriate patients [bib_ref] The importance of weight management in type 2 diabetes mellitus, Wilding [/bib_ref]. ## Research gaps While research and development efforts over the past few decades have led to the availability of several new classes of medications and new insulin formulations and delivery methods, we still lack a clear understanding of the ideal approaches to selecting appropriate treatment regimens for particular individuals. With a more in-depth characterization of the pathophysiology and natural history of subtypes of diabetes coupled with the pharmacogenomics of new and existing therapies, we can begin to develop a more personalized approach to diabetes management. Several areas can be immediately addressed. This includes performing clinical trials in vulnerable and understudied populations, including the elderly and children, that are critical to validate more precise evidence-based treatments in these populations. Studies examining the appropriate application of immune therapies in combination (sequentially or simultaneously) to target b-cell specific immune response, islet inflammation, and more global defective immunoregulation are critical. For type 2 diabetes, the early use of combinations of glucoselowering agents needs to be studied. For people with diabetes who are overweight or obese, studies are needed to determine whether weight loss medication and bariatric surgery could be used to support diabetes treatment goals. ## Complications Intensive glycemic control can reduce diabetes complications. In fact, in the decades since these studies were first published, rates of microvascular and macrovascular complications of diabetes and deaths from hyperglycemic crisis have substantially decreased [bib_ref] Changes in diabetes-related complications in the United States, Gregg [/bib_ref]. However, complications of diabetes remain the greatest health threat to people living with diabetes. Research efforts to identify clinical variables and biomarkers that indicate the presence or progression of complications may lead to a better understanding of risk and help identify individuals who may benefit from particular therapies to reduce the impact of diabetes. ## Type 1 diabetes The underlying pathophysiology driving an increased risk of cardiovascular complications in type 1 diabetes remains unclear. It is in part related to nephropathy and appears to be distinct from the pathophysiology of cardiovascular complications of type 2 diabetes [bib_ref] Type 1 diabetes mellitus and cardiovascular disease: a scientific statement from the, De Ferranti [/bib_ref]. Intensive treatment of type 1 diabetes with insulin often leads to weight gain. Concurrent with the population-wide rise in incidence of obesity, many people with type 1 diabetes have begun to exhibit features of obesity and metabolic syndrome, likely increasing the development of cardiovascular disease. Current treatment recommendations for management of cardiovascular risk factors predominantly derive from studies on type 2 diabetes or populations that did not discriminate between diabetes type. Risk factors should be monitored and treated in type 1 diabetes to recommended targets, but research is needed to determine distinctions in cardiovascular risk pathophysiology in type 1 diabetes and to identify appropriate therapies to reduce risk. Kidney disease predicts cardiovascular disease in people with type 1 diabetesand is associated with development of additional microvascular and macrovascular complications over time. People with type 1 diabetes show signs of premature arterial stiffening that is further exaggerated in those with diabetic nephropathy. There is a genetic propensity for diabetic nephropathy that peaks at 10-14 years duration of type 1 diabetes (158). The risk plateaus after 15 years duration, and the incidence of microalbuminuria matches this pattern (FinnDiane Study Group, unpublished observations). The peak incidence of macroalbuminuria and end-stage kidney disease lags 10 to 15 years behind the appearance of microalbuminuria. Progression to end-stage kidney disease is linked to age of onset and duration of diabetes [bib_ref] Incidence of end-stage renal disease in patients with type 1 diabetes, Finne [/bib_ref]. Female sex seems to be protective if age of onset occurs during or after puberty. Similar factors influence risk for and progression of diabetic retinopathy. Intensive glucose control significantly reduces the risk of diabetic peripheral neuropathy and cardiovascular autonomic neuropathy in type 1 diabetes. Average HbA 1c and HbA 1c variability are higher in people who progress to diabetic kidney disease. Those with more components of metabolic syndrome have more kidney disease and higher HbA 1c . A person with type 1 diabetes is much more likely to develop diabetic kidney disease if a sibling with type 1 diabetes has it. The risk of diabetic nephropathy in type 1 diabetes is fourfold higher in children whose mothers have type 1 diabetes than in those without a parent with diabetes (162), indicating a role for epigenetics in the development of kidney disease. Urine metabolites have been identified that highlight potential involvement of mitochondrial dysfunction in diabetic kidney disease [bib_ref] Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease, Sharma [/bib_ref]. ## Type 2 diabetes A large proportion of people with type 2 diabetes also have nonhyperglycemic components of the metabolic syndrome [bib_ref] The metabolic syndrome, Eckel [/bib_ref] , including hypertension, hyperlipidemia, and increased risk for cardiovascular disease. These metabolic features are interrelated and must be considered collectively. Multiple risk factor reduction is critical. Lipoprotein metabolism is often abnormal in diabetic nephropathy, but treatment strategies to avoid cardiovascular disease in this population are unclear. Statins appear to be ineffective at preventing cardiovascular disease in people with endstage kidney diease [bib_ref] AURORA Study Group. Cardiovascular disease in patients with renal disease: the role..., Fellström [/bib_ref] [bib_ref] German Diabetes and Dialysis Study Investigators. Atorvastatin in patients with type 2..., Wanner [/bib_ref]. Once on statins, fibrates may not be beneficial for preventing cardiovascular disease in this population but might have microvascular benefits through anti-inflammatory actions [bib_ref] Is fenofibrate a reasonable treatment for diabetic microvascular disease?, Simó [/bib_ref]. There are reasonably good data indicating that cholesterol absorption is higher in diabetes, suggesting that ezetimibe might have unique effects in diabetes [bib_ref] SHARP Investigators. The effects of lowering LDL cholesterol with simvastatin plus ezetimibe..., Baigent [/bib_ref] [bib_ref] IMPROVE-IT Investigators. Ezetimibe added to statin therapy after acute coronary syndromes, Cannon [/bib_ref]. Cardiovascular disease risk increases substantially when estimated glomerular filtration rate falls below 45 mL/min/1.73 m 2 . Microalbuminuria is not always due to diabetic nephropathy, but it is a marker of inflammation that indicates vascular leakage and increased cardiovascular risk. Albuminuria has been used as a marker of diabetic nephropathy for three decades. Yet, its power is limited. It varies by 25-30% daily in individuals [bib_ref] Should proteinuria reduction be the criterion for antihypertensive drug selection for patients..., Kalaitzidis [/bib_ref] [bib_ref] Editorial perspective. Should microalbuminuria ever be considered as a renal endpoint in..., Weir [/bib_ref] [bib_ref] Debate: CON position. Should microalbuminuria ever be considered as a renal endpoint..., Glassock [/bib_ref] [bib_ref] Microalbuminuria as a risk predictor in diabetes: the continuing saga, Bakris [/bib_ref]. It is transient and patients can revert to normal albuminuria without treatment. Interestingly, the urinary metabolomics signature of diabetic kidney disease is similar in people with type 1 and type 2 diabetes [bib_ref] Metabolomics reveals signature of mitochondrial dysfunction in diabetic kidney disease, Sharma [/bib_ref]. Newly identified biomarkers such as urinary adiponectin and serum tumor necrosis factor-a receptor 1 may be better predictors of nephropathy than albumin excretion rate; however, they require greater evaluation in prospective studies. Tight glycemic control is the only strategy known to prevent or delay the development of peripheral neuropathy, and cardiac autonomic neuropathy is perhaps even more important in relation to cardiovascular mortality [bib_ref] Diabetic cardiac autonomic neuropathy: Do we have any treatment perspectives?, Serhiyenko [/bib_ref]. However, randomized clinical trials to determine appropriate targets are lacking. Outcomes for cardiovascular disease and mortality have been mixed in different studies. ## Research gaps The assembled experts agreed that the means to determine which individuals with diabetes will develop particular complications remain unclear. Research efforts are needed to delineate the mechanisms underpinning the development of complications in type 1 diabetes and type 2 diabetes and identifying the differences between them. For example, the contributions of genetics to development of complications in specific populations need to be determined. The benefits of screening and early treatment to control glucose levels in people with presymptomatic diabetes on the development of complications also needs to be assessed. In some cases, the data supporting current treatment recommendations are drawn from populations that are too heterogeneous to be sufficiently representative of subtypes of diabetes. For example, current treatment recommendations for management of cardiovascular complications derive predominantly from data in type 2 diabetes or in populations that did not discriminate between diabetes type. Thus, data to support evidence-based targets to avoid cardiovascular complications in type 1 diabetes are needed. There are also some targeted issues that need to be addressed around specific complications to better inform treatment. For example, because of inconclusive associations, trials are needed to determine whether fibrates are able to modify the natural history of retinopathy and, if so, by what mechanisms. Given the limitations of current predictors of kidney disease progression, better biomarkers are needed. Finally, a better understanding of how complications of diabetes affect one another and how they impact treatment approaches is needed. This underlines a need for studies comparing the effectiveness of different strategies for glucose control in subpopulations with comorbidities. # Conclusions Diabetes is currently broadly classified as type 1, type 2, gestational, and a group of "other specific syndromes." However, increasing evidence suggests that there are populations of individuals within these broad categories that have subtypes of disease with a well-defined etiology that may be clinically characterized (e.g., LADA, MODY). These developments suggest that perhaps, with more focused research in critical areas, we are approaching a point where it would be possible to categorize diabetes in a more precise manner that can inform individual treatment decisions. Characterization of disease progression is much more developed for type 1 diabetes than for type 2 diabetes. Studies of first-degree relatives of people with type 1 diabetes suggest that persistent presence of two or more autoantibodies is an almost certain predictor of clinical hyperglycemia and diabetes. The rate of progression depends on the age of antibody onset, the number of antibodies, antibody specificity, and titer. Rising glucose and HbA 1c levels substantially precede the clinical onset of diabetes, making diagnosis feasible well before the onset of diabetic ketoacidosis. Three distinct stages of type 1 diabetes can be identified [fig_ref] Table 1 -: Staging of type 1 diabetes Fasting plasma glucose 100-125 mg/dL c 2-h... [/fig_ref] and serve as a framework for future research and regulatory decision-making [bib_ref] Staging presymptomatic type 1 diabetes: a scientific statement of JDRF, the, Insel [/bib_ref]. The paths to b-cell demise and dysfunction are less well defined, but deficient b-cell insulin secretion in the face of hyperglycemia appears to be the common denominator. Future classification schemes for diabetes will likely focus on the pathophysiology of the underlying b-cell dysfunction and the stage of disease as indicated by glucose status (normal, impaired, or diabetes). Recently, the All New Diabetics in Scania (ANDIS) study reported five distinct subtypes of diabetes on the basis of clustering of clinical, blood-based, and genetic information in newly diagnosed patients in Sweden. Importantly, these subtypes of diabetes appear to be differentially linked to risk for particular complications. The researchers confirmed similar groupings and relationships among patients in Finland. This model represents a notable example of an approach that, with additional information, could be refined in more diverse populations to begin developing meaningful classifications based on clinical characteristics, demographics, and novel biomarkers for disease risk, progression, and complications in discreet populations. Remaining critical research gaps are currently preventing the realization of true precision medicine for people with diabetes. The authors have outlined some of these key gaps [fig_ref] Table 1 -: Staging of type 1 diabetes Fasting plasma glucose 100-125 mg/dL c 2-h... [/fig_ref] and call for the diabetes research community to address these open questions to better understand genetic and molecular mechanisms of diabetes and its complications, define phenotypes and genotypes of subtypes of diabetes, and use this understanding in the development and application of therapies to prevent and treat diabetes and complications. Understanding the pathways to loss of b-cell mass and function is key to addressing all forms of diabetes and avoiding complications of diabetes; therefore, the gaps in these topic areas are highlighted as particular priorities among the many critical areas that remain to be investigated. By addressing the noted research gaps, we will be able to further refine models and make meaningful distinctions to stage diabetes. [fig] Figure: 1-Genetic and environmental risk factors impact inflammation, autoimmunity, and metabolic stress. These states affect b-cell mass and/or function such that insulin levels are eventually unable to respond sufficiently to insulin demands, leading to hyperglycemia levels sufficient to diagnose diabetes. In some cases, genetic and environmental risk factors and gene-environment interactions can directly impact b-cell mass and/or function. Regardless of the pathophysiology of diabetes, chronic high blood glucose levels are associated with microvascular and macrovascular complications that increase morbidity and mortality for people with diabetes. This model positions b-cell destruction and/or dysfunction as the necessary common factor to all forms of diabetes. [/fig] [table] Table 1 -: Staging of type 1 diabetes Fasting plasma glucose 100-125 mg/dL c 2-h glasma glucose 140-199 mg/dL c HbA 1c 5.7-6.4% or $10% increase in HbA 1c [/table]

Engaging community pharmacists to eliminate inadvertent doping in sports: A study of their knowledge on doping This study aimed to evaluate the community pharmacists' knowledge of tackling the issue of inadvertent doping in Malaysia. A cross-sectional survey was conducted among 384 community pharmacists working in Malaysia using a self-administered questionnaire. All the respondents were pharmacists fully registered with the Pharmacy Board of Malaysia and had been working in the community setting for at least one year. Of the 426 community pharmacists approached, 384 community pharmacists participated in this study, giving a response rate of 90.14%. The majority of the respondents were females (63.5%), graduated from local universities (74.9%), with median years of practising as a community pharmacist of six years (interquartile range, IQR = 9 years). The respondents were found to have moderate levels of doping-related knowledge (median score of 52 out of 100). Anabolic steroids (95.8%), stimulants (78.6%) and growth factors (65.6%) were recognised as prohibited substances by most of the respondents. Around 65.9% did not recognise that inadvertent doping is also considered a doping violation. Most of them (90%) also have poor levels of knowledge of doping scenarios in the country. Community pharmacists in Malaysia have limited knowledge in the field of doping. More programmes and activities related to doping and drugs in sports should be held to enhance the community pharmacists' knowledge on the issue of inadvertent doping. # Introduction Inadvertent doping is an issue where an athlete records a positive drug test after having unintentionally and unknowingly taken a banned substance [bib_ref] Inadvertent doping through supplement use by athletes: Assessment and management of the..., Baylis [/bib_ref]. Athletes may become ill or injured, a1111111111 a1111111111 a1111111111 a1111111111 a1111111111 or suffer from chronic medical conditions [bib_ref] Between medical treatment and performance enhancement: an investigation of how elite athletes..., Overbye [/bib_ref] , which may necessitate the use of medications that are banned in sports. For instance, in Japan, many over-the-counter medications contain prohibited substances; this explains the high rate of unintentional doping in the country [bib_ref] Knowledge of pharmacy students about doping, and the need for doping education:..., Shibata [/bib_ref]. A well-known case of inadvertent doping caused by using over-the-counter medications was at the 2000 Olympic Games. Pseudoephedrine, a banned substance, was mistakenly given to a Romanian gymnast by her team physician to treat her cold symptoms. This resulted in her being stripped of the gold medal that she won, and the team physician was expelled from the Games [bib_ref] Drug use in sports: a veritable arena for pharmacists, Ambrose [/bib_ref]. Some successful Malaysian badminton players, weightlifters, and wushu athletes were also caught in doping scandals, believed to be caused by inadvertent use of banned substances [bib_ref] Three causes of doping among athletes, Bernama [/bib_ref]. Inadvertent doping also involves the use of nutritional supplements among athletes. In past studies in Malaysia, 70% of the elite athletes and 40% of the youth athletes were reported to be consuming nutritional supplements [bib_ref] Knowledge and Attitude Related to Nutritional Supplements and Risk of Doping among..., Balaravi [/bib_ref] [bib_ref] Knowledge, attitude and practice on doping of Malaysian student athletes, Chiang [/bib_ref]. Inadvertent doping results when supplement labels contain misinformation that misleads athletes to falsely believe that prohibited products are allowed in sports [bib_ref] Nutritional Knowledge and Attitudes Towards Healthy Eating of College-Going Women Hockey Players, Davar [/bib_ref]. The key to addressing the issue of inadvertent doping by athletes is prevention . Pharmacist, as a healthcare professional that is specifically trained in medication use, can play an important role in combating doping in sports. Athletes may attend to the community pharmacy as a customer or a patient, to purchase medications, over-the-counter medications, or health supplements. For examples, prescribed medication such as salbutamol inhaler is a common medication used for asthma but excessive use without knowing the upper dose limit could violate the doping rules. Some over-the-counter medications may be sold with different active ingredients in different countries. A British skier had his Olympics bronze medal stripped due to unaware of the presence of banned substance in the formulation of the nasal inhaler he bought overseas during competition . Previous literatures also reported that some athletes obtained drug products from pharmacies while others would seek pharmacists' advice on the use of medications or supplements for various conditions, including the management of sports injuries [bib_ref] Perspectives of athletes and pharmacists on pharmacist-provided sports supplement counseling: An exploratory..., Howard [/bib_ref]. The International Pharmaceutical Federation (FIP) also recognises pharmacists' roles in preventing doping in sports by updating themselves on the World Anti-Doping Code and help athletes to identify prohibited substances in sports [bib_ref] Knowledge and Perceptions of Pharmacy Students in Qatar on Anti-Doping in Sports..., Awaisu [/bib_ref]. Although pharmacists are generally well-equipped with knowledge on medications, their abilities and readiness to counsel the athletes are yet to be known. The additional knowledge on the Prohibited List published by World Anti-Doping Agency (WADA) annually is the minimum requirement for the pharmacists to provide correct information to the athletes. Nevertheless, previous studies have reported that most of the pharmacists did not have sufficient knowledge on the prohibited substances in sports [bib_ref] Doping Knowledge, Attitude and Practice of Pharmacists in Dessie, Gebregergs [/bib_ref]. However, the current levels of knowledge among Malaysian community pharmacists about doping in sports are not well studied. This study expands on the survey conducted by Chiang et al in the capital city of Malaysia, Kuala Lumpur [bib_ref] The readiness of community pharmacists as counsellors for athletes in addressing issues..., Chiang [/bib_ref]. In our study, we have expanded the data collection to other states in Malaysia and assessed other aspects that were not investigated. Thus, this study aims to offer important insights into the factors that could influence community pharmacists' levels of doping-related knowledge and provides overview of Malaysian community pharmacists' knowledge of doping in sports especially with respect to their readiness to take part in anti-doping initiatives. Assessing their knowledge helps to identify new avenues for future studies and also areas of deficiency that would require interventions to improve pharmacists' current roles in assisting athletes with medication use and avoiding unintentional intake of banned substances. # Methods ## Study design and sampling This is a cross-sectional survey conducted online and via distribution of hard copies of questionnaires to community pharmacists in Malaysia. A list of registered community pharmacists was obtained from the Malaysian Pharmaceutical Society's website, and a simple randomised list was generated. The respondents included in this study were pharmacists fully registered with the Pharmacy Board of Malaysia and had been working in the community pharmacy setting for at least one year. ## Data collection A pilot study was carried out to test the reliability and validity of the questionnaire. The pilot study was done on 31 community pharmacists prior to data collection. Minor improvement was made based on their feedback. Questionnaires were distributed to selected community pharmacists from May 2019 until November 2019. They were briefed about the objectives of the study before consenting to take part. We have included an explanatory statement at the beginning of the questionnaire that advises the respondents not to refer to any resources when answering the knowledge-based questions. This study was approved by the Research Ethics Committee, the National University of Malaysia (UKM PPI/111/8/JEP-2018-215). ## Sample size calculation To ensure that the study findings generalizable to the whole community pharmacist population in Malaysia, we used the Krejcie and Morgan formula was used to calculate the required sample size [bib_ref] Determining sample size for research activities, Krejcie [/bib_ref]. The calculation was based on the total number of community pharmacists in Malaysia, determined to be 3094. The minimum sample size was calculated at 384. ## Study instruments The demographic section contained 13 questions about socio-demographic characteristics, namely gender, age, race, place of practice, type of place of practice, practice premises, profession, academic qualification, experience of practising abroad, postgraduate qualification, professional membership, number of years in practice, and understanding of the term 'doping'. We took into accounts of the experience of practicing abroad as improved knowledge on pharmacy practice and patient care is associated with exposure of international experiences [bib_ref] Global Health Learning Outcomes by Country Location and Duration for International Experiences, Steeb [/bib_ref]. The knowledge section included questions that were adapted from a previous survey that assessed the readiness of community pharmacists as counsellors for athletes [bib_ref] The readiness of community pharmacists as counsellors for athletes in addressing issues..., Chiang [/bib_ref]. The questions can be classified into five main domains: 1) Prohibited substances in sports; 2) The roles of WADA; 3) Anti-doping rule violations; 4) General knowledge of doping; 5) Doping cases in Malaysia. We wrote several additional questions about the roles of WADA, types of doping violations, athlete biological passport (ABP), and the doping situation in Malaysia, based on The Prohibited List 2019 and World Anti-Doping Code 2019 [bib_ref] Doping awareness, views, and experience: a comparison between general practitioners and pharmacists, Auersperger [/bib_ref]. Overall, this section consists of 10 multiple-choice and true or false questions. Assessment of the knowledge is done based on the marks that the respondents obtain. A score of 2 was given to each correct answer; 1 for 'not sure'; and 0 for each wrong answer. The total score was 68. The respondents were then grouped into three categories based on their knowledge levels. Respondents who scored 41 or less (60% or less of 68) were classified as having poor levels of knowledge; 42-56 (61%-83% of 68) as having moderate levels of knowledge; and 57 or more (84% or more of 68) as having good levels of knowledge [bib_ref] The readiness of community pharmacists as counsellors for athletes in addressing issues..., Chiang [/bib_ref] [bib_ref] Doping awareness, views, and experience: a comparison between general practitioners and pharmacists, Auersperger [/bib_ref]. # Data analysis The data were analysed using the Statistical Package of Science Analysis (SPSS) version 25. Continuous data was presented as medians alongside values for the interquartile range. The Mann-Whitney U and Kruskal-Wallis tests were used to determine the association between the respondents' demographic characteristics and knowledge scores. A p-value of <0.05 was used to mark statistical significance. # Results ## Pilot study The reliability and validity of the questionnaire was assessed (Npilot = 31) prior to data collection. The value of Cronbach's alpha obtained for the knowledge section was 0.780, which indicated good reliability. ## Response rate Over a period of seven months, 426 questionnaires were distributed, and 384 questionnaires were completed and returned, giving a response rate of 90.14%. Overall, the missing value calculated from the valid responses was 0.61%. ## Demographic characteristics The demographic characteristics of the respondents (N = 384) are shown in [fig_ref] Table 1: The respondents' demographic characteristics [/fig_ref] The majority of the respondents (N = 284, 74.9%) obtained their pharmacy degrees from local universities. Only 13.1% (N = 50) of the respondents had worked abroad, and most of them did so for less than one year (N = 25, 6.5%). A small number (4.2%, N = 16) of the respondents had postgraduate degrees (PhD or master's). Most of the respondents (N = 248, 65.6%) were members of professional bodies, the main one being the Malaysian Pharmaceutical Society (MPS) (N = 241, 62.8%). In terms of their professional experience measured by years in practice, most of the respondents had been practising in various settings for a total of two to five years (N = 147, 39.2%). The median number of years of working as a community pharmacist was six years with the interquartile range (IQR) being nine years. ## The respondents' familiarity of the term 'doping' More than three-quarters of the respondents (N = 307, 80.6%) had heard of the term 'doping'. shows a descriptive analysis of the five domains of the respondents' knowledge of doping in sports. ## The respondents' knowledge of doping in sports Knowledge of prohibited substances in sports. Most of the respondents knew that anabolic-androgenic steroids (N = 368, 95.8%), stimulants (N = 302, 78.6%), and growth factors (N = 252, 65.6%) are prohibited in sports. These drugs are prohibited at all times in sports. Insulin (N = 31, 8.1%) and beta-blockers (N = 98, 25.5%) were, however, lesser known among the respondents. Alcohol (N = 78, 20.3%) was most frequently mistaken by the respondents as a prohibited substance, followed by caffeine (N = 68, 17.7%), nicotine (N = 58, 15.1%) and NSAIDs (N = 33, 8.6%). We found that 71.6% of the respondents (N = 275) were aware that This section also contains questions about the use of drugs in competition. Most of the respondents (N = 272, 70.8%) were able to identify salbutamol as being allowed in competition, but less than half of the respondents were able to do so for salmeterol (N = 172, 44.8%) and inhaled corticosteroids (N = 175, 45.6%). Less than a quarter of the respondents misidentified oral corticosteroids (N = 46, 12.0%), injected corticosteroids (N = 35, 9.1%), and dihydrocodeine (N = 18, 4.7%) as substances allowed in competition. Knowledge of the roles of the World Anti-Doping Agency (WADA). The role of WADA in coordinating anti-doping initiatives worldwide was correctly identified by most of the respondents (N = 314, 81.8%), and this is followed by the other functions the agency serves in publishing updated lists of prohibited substances in sports (N = 293, 76.3%) and establishing the World Anti-Doping Code (N = 251, 65.4%). Most of the respondents (N = 254, 66.1%) knew that WADA does not prosecute athletes who violate anti-doping rules. However, many of them (N = 246, 64.1%) did not know that the tests for detecting prohibited substances in blood or urine samples are not conducted by WADA but WADA-accredited laboratories. Knowledge of doping violations. Most of the respondents were aware that doping violations include the presence of a prohibited substance in a blood or urine sample (N = 352, 91.7%), refusal to undergo a doping test requested by authorised personnel (N = 286, 74.5%), and administration of a prohibited substance to an athlete (N = 276, 71.9%). Slightly over half of the respondents (N = 196, 51.0%) were aware that being complicit in the trafficking of prohibited substances to athletes is also a doping violation. Only a minority of the respondents knew that unintentional intake of prohibited substances by athletes is a doping offense (N = 131, 34.1%). General knowledge of doping. More than half of the respondents (N = 243, 63.3%) knew that the Therapeutic Use Exemption (TUE) allows athletes to use prohibited substances for medical reasons in or out of competition. We found that only 26.0% (N = 100) correctly identified ABP as a programme that monitors selected biological variables over time to indirectly reveal the effects of doping. Knowledge of the anti-doping initiatives in Malaysia. Most of the respondents (N = 364, 94.8%) mistakenly assumed or were unsure whether the professional bodies in Malaysia provided guidelines on the use of prohibited substances in sports. Many of the respondents (N = 376, 97.9%) were still unaware that the Analytical Biochemistry Research Centre, Universiti Sains Malaysia had been removed from WADA's list of accredited laboratories that carry out anti-doping drug testing. Also, a small number of respondents (N = 42, 10.9%) knew that the National Sports Institute in Malaysia is not the official anti-doping agency in Malaysia. Total scores of the respondents' doping-related knowledge. Based on their total knowledge scores, the respondents were classified as having poor, moderate, or good levels of knowledge of doping in sports [fig_ref] Table 3: The respondents' levels of knowledge of doping [/fig_ref]. Only 1.8% (N = 7) of the respondents scored 41 or less (poor levels of knowledge). More than three-quarters of the respondents (N = 317, 82.6%) scored between 42 to 56 (moderate levels of knowledge). A quarter of the respondents (N = 60, 15.6%) scored 57 or more (good levels of knowledge). The median score was 52 with an IQR of 6. shows the relationship between the respondents' demographic characteristics and knowledge scores. ## Statistical studies The respondents were grouped based on their demographic characteristics. The Mann-Whitney U test (U value) was used when the comparison of mean ranks (knowledge scores) involved only two groups of respondents, while the Kruskal-Wallis test (H value) was used to compare mean ranks between more than two groups of respondents. Through these statistical analyses, we found the knowledge scores to be significantly affected by the respondents' postgraduate qualifications (or lack thereof) (p = 0.047), professional membership (p = 0.001), amount of professional experience measured in years (p = 0.045), and understanding of the term 'doping' (p<0.0001). As shown in [fig_ref] Table 5: Comparison of mean rank between associated demographic variables and total score of... [/fig_ref] # Discussion The current study evaluated the Malaysian community pharmacists' knowledge related to drugs in sports. In general, most of the respondents had heard of the term 'doping' and were able to describe it adequately as the use or misuse of drugs by athletes to enhance their performance in sports. Most of them were able to identify anabolic-androgenic steroids (AAS), stimulants, and growth factors as prohibited substances in sports. This is consistent with previous studies, which reported that pharmacists were able to identify anabolic-androgenic steroids and stimulants as prohibited substances in sports [bib_ref] Retail pharmacists and doping in sports: Knowledge and attitudes. A national survey..., Laure [/bib_ref] [bib_ref] Perspective of pharmacists in Qatar regarding doping and anti-doping in sports, Mottram [/bib_ref]. We also found that most of the respondents knew that diuretics could be used as masking agents, a finding similarly reported by Chiang et al [bib_ref] The readiness of community pharmacists as counsellors for athletes in addressing issues..., Chiang [/bib_ref]. The pharmacists' familiarity with these substances could be associated with the popularity of the substances in doping cases and they were the most abused substances in sports as proven by the reports by WADA in 2017 stating that up to 58% of all adverse analytical findings in doping tests came from AAS and stimulants. However, most of the respondents failed to identify insulin and beta-blockers as prohibited substances. This is because beta blockers are prohibited in-competition for certain sports only. So, the respondents might have lesser awareness on these unpopular doping substances. Additionally, WADA reported in 2017 that only 0.3% of doping tests were positive for betablockers, suggesting that the drugs were infrequently misused by athletes. This may explain why these substances were less recognised by the respondents as prohibited substances. Meanwhile, the respondents might not be aware of the mechanism and the reason of insulin being used as a doping agent. Insulin is normally used by diabetic patients for treating high sugar level, but it could be misused by bodybuilders and weightlifters to suppress proteolysis and increase protein synthesis for faster muscle gain [bib_ref] Insulin as a drug of abuse in body building, Evans [/bib_ref]. Besides, the study extends our knowledge on the familiarity of the Malaysian community pharmacists on the definition of doping violations. Most of the respondents in this study were unaware that helping in trafficking prohibited substances to athletes and unintentional intake of a prohibited substance are also considered doping violations. The results show the lack of awareness of the community pharmacists on the doping definition published by WADA which clearly states that athletes should be responsible for everything they ingest, and even accidental intake of banned substance would violate the doping rules. Therefore, pharmacists need to step up in expanding their knowledge so that in the future they could advise the athletes and become their support personnel in building a healthy and sustainable sports career for them. Only 63.3% of the respondents in this study knew that TUEs are required for the use of drugs by athletes, and the proportion is lower than the 75.9% reported by Chiang et al [bib_ref] The readiness of community pharmacists as counsellors for athletes in addressing issues..., Chiang [/bib_ref]. In comparison, 45.2% of the South African pharmacists who responded to a survey scored poorly on the knowledge regarding TUEs [bib_ref] Anti-Doping Knowledge and Opinions of South African Pharmacists and General Practitioners, Starzak [/bib_ref]. These findings indicate that there is still a need for educating pharmacists on the importance of TUEs as a mechanism that enables the use of prohibited substances or methods in the treatments of illnesses, injuries, or chronic medical conditions experienced by athletes [bib_ref] Therapeutic Use Exemptions, Gerrard [/bib_ref]. Most of the respondents in this study were also unaware that ABP is a newly introduced doping detection method. ABP is relatively simple and can be potentially adopted by many countries as an effective measure against doping [bib_ref] The athlete biological passport, Sottas [/bib_ref]. A full understanding of the harmonised modules employed in ABP, including the haematological and steroidal modules, is the first step towards establishing proper ABP testing facilities. The ABP Operating Guidelines, published by WADA, harmonize both modules and are a good resource for establishing proper ABP testing facilities. Furthermore, almost 90% of the respondents in our study did not know the doping initiatives and official bodies in Malaysia. Malaysia once had an accredited laboratory at Universiti Sains Malaysia, which was suspended due to non-compliance with the International Standard for Laboratories . Besides, most of the community pharmacists did not know that the official anti-doping agency in Malaysia that is tasked with fighting doping is Anti-Doping Agency of Malaysia (ADAMAS) despite its existence since 2007. The percentage is substantially lower than that reported in another study i.e., 54.9% of Slovenian pharmacists knew their national anti-doping agency [bib_ref] Doping awareness, views, and experience: a comparison between general practitioners and pharmacists, Auersperger [/bib_ref]. Failure to recognize the proper source of information to refer to when meeting athletes in their working environment may make pharmacists unable to provide correct recommendations and advice to the athletes. The Irish College General Practitioners (ICGP) published guidelines to educate general practitioners on doping-related regulations and their roles and involvement in the prevention of doping in sports. The guidelines are reviewed periodically, with the latest edition being published in 2015. In contrast, no professional bodies in Malaysia have published guidelines on the use of prohibited substances in sports; but most of the respondents in our study were unaware of this. Professional bodies in every country, including Malaysia, should adopt a similar practice to ICGP and publish guidelines for engaging healthcare professionals in the prevention of doping in sports. Overall, our study demonstrated that the average knowledge score of Malaysian community pharmacist on doping was moderate. This is in line with previous literatures by [bib_ref] Anti-doping knowledge and educational needs of Finnish pharmacists, Lemettilä [/bib_ref] and Gebregers et al (2021) [bib_ref] Doping Knowledge, Attitude and Practice of Pharmacists in Dessie, Gebregergs [/bib_ref] [bib_ref] Anti-doping knowledge and educational needs of Finnish pharmacists, Lemettilä [/bib_ref]. These findings pointed out the needs to improve pharmacists' knowledge in drugs in sports which could be done by establishing courses on drugs in sports during university study or special courses on drugs in sports for the working pharmacists. In Malaysia, subjects related to doping in sports are incorporated in curriculum in pharmacy programmes either as a core subject with three credit hours or elective subject with two credit hours. However, some universities in Malaysia did not offer the subjects to their students [bib_ref] Knowledge, attitude and perception of Malaysian pharmacy students towards doping in sports, Yuen [/bib_ref]. The lack of exposure and training provided during university may then cause the pharmacists to have low confidence when they are dealing with issues related to prohibited substances in sports. The correlation analysis showed that the respondents with six or more years of professional experience had better knowledge and could potentially be trained to become drug advisors or counsellors for athletes. A good understanding of the term 'doping' predicted better knowledge scores. Thus, pharmacists should be encouraged to learn more about doping-related issues. We found that the respondents who were members of professional bodies obtained significantly better knowledge scores. Most of the respondents in this study were members of Malaysian Pharmaceutical Society. This national association for pharmacists periodically organises courses and seminars for professional development, averaging~15 programmes per month in the past three years. Thus, pharmacists that are interested in getting more information on drugs in sports should take self-initiative to attend relevant courses offered by antidoping agency to keep themselves up to date. Sports pharmacy is considered a relatively new and emerging fields especially in South-East Asia. Pharmacists are traditionally perceived by the public as specialists in medication dispensing and counselling. However, their role of pharmacist in healthcare has expanded over the years towards primary prevention through health education. Athletes are a special population with generally good health status but may still consume a relatively large number of medications and supplements compared to the ordinary healthy individuals. Thus, it is important for pharmacists to engage and provide their professional service to athletes in the future to eliminate inadvertent doping. # Limitations The first limitation of the study relates to the use of self-administered questionnaires. Although the explanatory statement clearly indicates that the respondents should answer the knowledgebased questions honestly without referring to any resources, some might not have followed the guidelines. This may have led to inaccuracy in the assessment of the respondents' knowledge. Second, the respondents were recruited through convenience sampling, and the questionnaires were not evenly distributed to community pharmacists in the different states of Malaysia. Thus, the results of this study may not be generalised to the entire population of community pharmacists across Malaysia. # Conclusion We found that community pharmacists in Malaysia had moderate levels of doping-related knowledge. They were able to identify prohibited substances commonly misused by athletes. Most were still unaware that inadvertent doping constitutes a doping violation, despite its being the primary contributor to the prevalence of doping in sports. Most were also illinformed about the doping situation in Malaysia. Hence, more doping-related programmes and activities should be organised to enhance community pharmacists' knowledge of inadvertent doping and transform them into proactive participants in contemporary anti-doping initiatives. Supporting information S1 [table] Table 1: The respondents' demographic characteristics (n = 384). [/table] [table] Table 3: The respondents' levels of knowledge of doping (N = 384). [/table] [table] Table 5: Comparison of mean rank between associated demographic variables and total score of community pharmacists' knowledge. [/table] [table] Table: Supporting information for respondents' demographic characteristics. (XLSX) S2Table. Supporting information for respondents' levels of knowledge in doping. (XLSX) S3 Table. Supporting information for demographic variables and the respondents' knowledge scores. (XLSX) 9. Reardon CL, Factor RM. Sport psychiatry: a systematic review of diagnosis and medical treatment of mental illness in athletes. Sports Med. 2010; 40(11):961-80. https://doi.org/10.2165/11536580-000000000-00000 PMID: 20942511 10. Los Angeles Times Archives. British Skier Stripped of His Olympic Medal; 2002. [cited 2022 Apr 4] Available from: https://www.latimes.com/archives/la-xpm-2002-mar-22-sp-oly22-story.html#:~:text= The%20International%20Olympic%20Committee's%20executive,at%20last%20month's%20Winter% 20Games. 11. Tsarouhas K, Kioukia-Fougia N, Papalexis P, Tsatsakis A, Kouretas D, Bacopoulou F, et al. Use of nutritional supplements contaminated with banned doping substances by recreational adolescent athletes in Athens, Greece. Food Chem Toxicol. 2018; 115:447-50. https://doi.org/10.1016/j.fct.2018.03. 043 PMID: 29621580 [/table]

Exploring experiences and needs of spousal carers of people with behavioural variant frontotemporal dementia (bvFTD) including those with familial FTD (fFTD): a qualitative study Introduction: Carers of people with frontotemporal dementia (FTD) experience greater challenges than carers of people with other dementias due to the younger age of onset and the challenging presentation of symptoms. The aim of the present study was to explore experiences of spousal carers of people with bvFTD, including those with the familial form of the disease (fFTD).Method: Fourteen qualitative interviews were analysed using an inductive approach to Thematic Analysis to understand experiences of spousal carers of people with bvFTD including those with fFTD. # Introduction Frontotemporal dementia (FTD) is the second most common cause of dementia in people under the age of 65 years [bib_ref] Frontotemporal Dementia: An Updated Clinicians Guide, Rosness [/bib_ref] , and is attributed to 25% of individuals with dementia in those over 65 [bib_ref] The epidemiology of frontotemporal dementia, Onyike [/bib_ref]. FTD is a neurodegenerative condition that impairs executive functioning and language, impacting on behaviour, planning, problemsolving, emotional control and speech [bib_ref] Frontotemporal dementia, Warren [/bib_ref]. FTD encompasses a behavioural variant (bvFTD) and a language variant, known as primary progressive aphasia (PPA) [bib_ref] Classification of primary progressive aphasia and its variants, Gorno-Tempini [/bib_ref] [bib_ref] Alzheimer's pathology in primary progressive aphasia, Rohrer [/bib_ref]. The language variant can be further subdivided into semantic (svPPA), non-fluent (nfvPPA) and logopenic variants (lvPPA), however lvPPA is often associated with underlying Alzheimer's disease pathology [bib_ref] Classification of primary progressive aphasia and its variants, Gorno-Tempini [/bib_ref] [bib_ref] Alzheimer's pathology in primary progressive aphasia, Rohrer [/bib_ref]. Cases with a family history (~ 30-40% of cases) are referred to as familial FTD (fFTD) [bib_ref] The heritability and genetics of frontotemporal lobar degeneration, Rohrer [/bib_ref]. The majority of fFTD is caused by a genetic mutation inherited in an autosomal dominant manner, usually in one of three genes, C9orf72, MAPT and GRN [bib_ref] The heritability and genetics of frontotemporal lobar degeneration, Rohrer [/bib_ref] , meaning that a child born to a parent with the disease has a 50% chance of inheriting the mutation [bib_ref] Clinical, genetic and pathological heterogeneity of frontotemporal dementia: A review, Seelaar [/bib_ref]. fFTD can present in all of the variants, but most people develop bvFTD, which is the focus of this study [bib_ref] The heritability and genetics of frontotemporal lobar degeneration, Rohrer [/bib_ref]. New diagnostic criteria for bvFTD were developed in 2011, when the International Behavioural Variant FTD Criteria Consortium (FTDC) used pathologically confirmed cases of FTD to create sensitive and specific clinical criteria for diagnosing bvFTD [bib_ref] Sensitivity of revised diagnostic criteria for the behavioural variant of frontotemporal dementia, Rascovsky [/bib_ref]. The criteria identified six behavioural and cognitive symptoms central to the diagnosis: i) apathy, ii) behavioural disinhibition, iii) loss of sympathy or empathy, iv) perseverative or compulsive behaviour, v) hyperorality and dietary changes, and vi) executive and language dysfunction. BvFTD diagnosis requires three of these symptoms to be present [bib_ref] Diagnosis and management of behavioral issues in frontotemporal dementia, Manoochehri [/bib_ref]. Despite these criteria, symptoms are often misdiagnosed by healthcare professionals as a psychiatric illness before reaching a diagnosis of bvFTD [bib_ref] Frontotemporal Dementia: An Updated Clinicians Guide, Rosness [/bib_ref] [bib_ref] Frontotemporal dementia, Warren [/bib_ref] [bib_ref] The Scale for Emotional Blunting in patients with frontotemporal dementia, Mendez [/bib_ref]. Carers of people with FTD (whether bvFTD or FTD in general) experience greater overall burden [bib_ref] Impact of behavioural problems on spousal caregivers: A comparison between Alzheimer's disease..., Vugt [/bib_ref] [bib_ref] Burden in carers of dementia patients Higher levels in carers of younger..., Freyne [/bib_ref] [bib_ref] The psychosocial impact of young onset dementia on spouses, Kaiser [/bib_ref] [bib_ref] Caregiver burden, healthrelated quality of life and coping in dementia caregivers: A..., Riedijk [/bib_ref] , stress and depression [bib_ref] Factors underlying caregiver stress in frontotemporal dementia and Alzheimer's disease, Mioshi [/bib_ref] than carers of people with typical, late-onset forms of dementia such as Alzheimer's disease. This is probably due in part to younger age of onset and the genetic link [bib_ref] Frontotemporal dementia, Warren [/bib_ref] , but it is the behavioural changes in FTD that are often reported as the most stressful by carers [bib_ref] Impact of behavioural problems on spousal caregivers: A comparison between Alzheimer's disease..., Vugt [/bib_ref] [bib_ref] Validation of the Addenbrooke's Cognitive Examination III in Frontotemporal Dementia and Alzheimer's..., Hsieh [/bib_ref] [bib_ref] Making sense of dementia and adjusting to loss: Psychological reactions to a..., Robinson [/bib_ref] [bib_ref] Family caregivers of individuals with frototemporal dementia, Wong [/bib_ref]. As a consequence of the disease, carers describe a loss of the interpersonal relationship with the person who was once seen as a companion, spouse, lover and friend [bib_ref] Caring for loved ones with frontotemporal degeneration: The lived experiences of spouses, Massimo [/bib_ref] [bib_ref] Relatives' experiences of frontal-variant frontotemporal dementia, Oyebode [/bib_ref] [bib_ref] Family caregivers experiences of the pre-diagnostic stage in frontotemporal dementia, Rasmussen [/bib_ref]. In the UK support for caregivers of people with dementia is largely provided within the community through primary care (general practitioners) and specialist memory services. As such much of this is designed for AD and older adults. Support for those with rare and early onset dementias is much less common. There is, therefore, significant geographical variation in services available to individuals within their community. There is a breadth of literature exploring the needs of carers of people with typical forms of dementia, such as Alzheimer's disease and vascular dementia. Interventions for these groups have shown promise in improving carer wellbeing and reducing burden associated with caring. However, it is unclear if these interventions (that have been developed to support carers of people living with dementia in general) are effective for carers of bvFTD. Furthermore, there are few studies exploring the subjective experiences and needs of family carers of people with bvFTD [bib_ref] Family caregivers of patients with frontotemporal dementia: An integrative review, Caceres [/bib_ref] and a small number of interventions piloted in caregivers of those with FTD, or both FTD and Alzheimer's disease together [bib_ref] Online information and support for carers of people with young-onset dementia: A..., Metcalfe [/bib_ref] [bib_ref] Improving Burden and Coping Skills in Frontotemporal Dementia Caregivers: A Pilot Study, Mioshi [/bib_ref] [bib_ref] Positive behaviour support in frontotemporal dementia: A pilot study, O&apos;connor [/bib_ref]. To our knowledge, there have been no randomised control trials and there are no specific interventions developed to support caregivers of people with behavioural variant FTD. This is particularly so in the area of fFTD where there may be implications for young families coping with the genetic version of the disease. It is important to consider the specific issues experienced by those caring for people with FTD as general dementia support services available within the community are often not appropriate. For example, the earlier age at onset often experienced in FTD can cause increased strain on caregivers who are often still in employment and may also have a family to take care of. Additionally, for those with fFTD, there is often the added issue of heritability, as caregivers also take on the burden of their children living at-risk of FTD. Despite an increase in research studies in the past decade to understand FTD pathology, genetic links, and prognosis, literature on understanding the psychosocial needs of families of people living with this debilitating disease is limited [bib_ref] Progress and Challenges in Frontotemporal Dementia Research: A 20-Year Review, Hodges [/bib_ref]. Few studies have employed a qualitative methodology, and the studies that are available have used very small sample sizes [bib_ref] Caring for loved ones with frontotemporal degeneration: The lived experiences of spouses, Massimo [/bib_ref] , and mixed multiple variants of FTD together when reporting their findings [bib_ref] The unique experience of spouses in early-onset dementia, Ducharme [/bib_ref]. These are limitations of the existing literature, and raises concerns regarding the transferability of its findings to carers of people with bvFTD and fFTD. The current research aims to identify the psychosocial support needs and experiences of spousal carers of people with bvFTD and fFTD. The analyses will identify barriers and facilitators of getting these needs met, and will highlight potential similarities and differences across a group of non-genetic (sporadic) bvFTD and a group with a genetic form of this subtype (bvFTD due to fFTD). # Method The current study took a phenomenological stance to understand the experiences of carers of people with bvFTD and fFTD, and explored their views, perceptions and experiences of being a spousal carer of a person with bvFTD, as well as the meaning they attach to these. ## Participants and setting Participants were 14 spousal carers living with a person diagnosed with bvFTD, five of whom were caring for people with sporadic FTD, and nine of whom were caring for people with fFTD. Inclusion criteria were: a) membership of an FTD support group, b) a main (Spousal) carer of a person with bvFTD. Carers with a diagnosis of a major psychiatric disorder, learning disability, or individuals unable to understand English, were excluded. Interviews were conducted three months to 11 years post diagnosis, with nine being diagnosed within one year of participating in the study. Characteristics of carers and care recipients are reported in . ## Recruitment and data collection Participants were identified from two existing UCL Queen Square Institute of Neurology Dementia Research Centre studies, which recruited participants from across the United Kingdom; the Longitudinal Investigation of FTD (LIFTD), and the GENetic Frontotemporal dementia Initiative (GENFI). The sample was initially recruited to have an even split between carers of people with sporadic and familial bvFTD. However, a more opportunistic sampling was later used to reach thematic saturation, with a sample of 14 participants recruited over a period of seven months. Participants were provided with information regarding the interview study by CG either verbally or via email. Interviews were conducted in person (n = 5), via Skype (n = 8) or telephone (n = 1), depending on participant preference. Written informed consent and ethical approval was obtained. Participant confidentiality was maintained by assigning pseudonyms. In-depth, semi-structured interviews were conducted in order to explore carers' support needs and experiences of being carers of people with bvFTD and fFTD. Development of the interview schedule was informed by a literature review [bib_ref] Improving Burden and Coping Skills in Frontotemporal Dementia Caregivers: A Pilot Study, Mioshi [/bib_ref] and developed in collaboration with the LIFTD and GENFI research teams and piloting with a bvFTD carer. The expert by experience pilot interview was not included in the final sample. The interview explored the impact of FTD on participants' lives, their positive and negative experiences, and explored their support needs (see Appendix A for the interview schedule). Interviews were conducted by an experienced qualitative researcher (ST), ranged in length between 45-90 min and took place between April and November 2019. All interviews were audiorecorded and transcribed verbatim. The researcher (ST) explored and attended to preconceptions and biases prior to the analysis with the aim of avoiding interference with data collection and interpretation. # Analysis Interviews were analysed using an inductive and reflexive approach to Thematic Analysis [bib_ref] Using thematic analysis in psychology, Braun [/bib_ref]. Patterns and themes across a dataset and the differences between sporadic and familial bvFTD were also the focus of the analysis. A systematic six-step process was followed according to the steps outlined for this method [bib_ref] Reflecting on reflexive thematic analysis, Braun [/bib_ref] and NVivo software (Version 12, for Mac) was used to aid in the process of coding and analysis. Analysis steps included: i) data familiarisation ii) initial coding iii) searching for themes by grouping codes iv) checking the themes v) defining and naming the themes vi) report writing. Initial development of themes was reviewed by the senior author. Thirteen participants were approached to review the write-up of the results, two participants responded and no changes were required as a result of this process. The remaining 14 th participant was not approached for review due to health issues and unavailability by email. # Results Five main themes were identified to depict key aspects of understanding the experiences and needs of spousal carers of people with FTD. Main themes included: (i) The "Constant Battle" -A journey toward an FTD diagnosis, (ii) Shock, Relief and Fear -Challenges persist post diagnosis, (iii) The "Life Altering" impact -The loss of the spousal relationship and shifting roles, (iv) Adapting, managing symptoms and receiving support, (v) Lack of general knowledge-Barriers to support for spousal carers of FTD. See for the representation of themes across participants. ## The "constant battle" -a journey toward an ftd diagnosis All of the carers expressed having been through a challenging road to receiving a diagnosis for their spouses' bvFTD. Challenging stages along this journey included: noticing slow and subtle symptoms, misattributing symptoms to situational, psychological or relationship problems, and feeling isolated in the experience leading up to diagnosis. The excerpt below demonstrates the experience of noticing the initial symptoms of FTD as subtle and gradual changes in the personality and behaviour of her spouse. Another carer outlined the process of trying to make sense of the initial changes she was observing with her husband's behaviour and attributing symptoms of aggression, impulsivity and loss of empathy to psychological or relationship difficulties in their marriage. In an attempt to make sense of symptoms carers sought out information that could help them to understand their partner's unusual and at times distressing behavioural and personality changes. During this process, carers described feeling socially isolated when family, friends or professionals did not take concerns seriously. ## C14 ## C7: i felt like i was completely on my own in saying, "it's not eccentricity. it's not him getting old. it's not stress"[…] so [before receiving the diagnosis] it was a constant battle with explaining for him what was going on. (female, fftd) Barriers experienced in medical services included initial misdiagnoses of other physical or psychiatric conditions and various mental health conditions. Carers Representation of Themes Across Participants described feeling as though concerns were not taken seriously by medical professionals, and that they needed to "bully" (C2) or push for further testing to reach an appropriate diagnosis. C7 then recounted having to further justify seeking a new diagnosis by acquiring information about the condition to inform the dementia services clinic about FTD, and advocate for her spouse to receive further specialist diagnostic testing. ## Shock, relief and fear -challenges persist post diagnosis Upon receipt of a FTD diagnosis, 12 carers reported experiencing mixed feelings of relief and fear about the future, and a continued sense of isolation, given the lack of support offered after receiving the diagnosis. ## C8: when he got the diagnosis, i wasn't happy, but i was relieved that i knew that there was something wrong with him and it wasn't my imagination. […] I kind of felt a bit naively, "Oh, this will be all right. We'll deal with this. I'll just sit in front of the TV and he'll sit there quietly and we'll get on." But I didn't realize with this disease that he's got, it doesn't work like that. I didn't really know what was going to happen. (Female, sporadic bvFTD) After receiving the diagnosis, some carers described feeling a sense of relief that they now had a label that could explain their spouse's behaviour. Out of the 12 carers who spoke about their experience of receiving a diagnosis, all but one of the carers (C3) expressed having continued difficulty coping with the uncertainty regarding managing expectations of their spouse's future following diagnosis. The life-altering impact -the loss of the spousal relationship and shifting roles All 14 participants made reference to the many life-altering aspects of becoming a spousal carer for a person with bvFTD. Participants described having difficulty managing caring duties alongside increasing responsibilities in the household, shifting roles, addressing their family's future risk, and experiencing a deep sense of loss as a result of no longer feeling able to connect or communicate with their spouse. C7: The main challenge-it's-you go from a family unit with two parents and, you know, doing everything together, to a single parent, which is one thing. But then it's a single parent with a parent-because there's somebody there, but you've got responsibilities, plus you've got someone else to look out for. (Female, fFTD) Carers additionally described the difficulties of managing responsibilities in the home and the emotional toll that their spouse's behavioural symptoms had on them. Eight carers described difficulties related to sleep as the most significant issue for them. Participants described feeling exhausted and having difficulties falling or staying asleep as a result of their spouse's inconsistent or interrupted sleeping behaviour. Carers described their own lack of sleep as the main contributor to frustration, lack of patience with their spouse and difficulty coping with caring responsibilities. Others reported that some of the care-recipients slept more than they used to, and this allowed them time for respite in the day, while one carer described oversleeping as an issue that interfered with her ability to share quality time with her spouse (C14). One of the experiences described by spousal carers as the most difficult was the experience of loss, described by seven participants, who expressed having difficulty coming to terms with the reality that they could no longer connect or communicate with their spouse or life partner. This created a deep sense of loss (for example of empathy and memory), with carers likening it to losing a life partner along with their previously held hopes and expectations for their future together. Four carers likened these difficulties in communicating and changing roles to that of caring for a child. However, they clarified this by stating that unlike children, their spouse had a diminishing capacity to learn and would become increasingly more dependent on them to meet their needs. Carers of spouses diagnosed with the familial form of bvFTD (fFTD) described the impact of identifying a strong genetic link and the implications this holds for both the carer and the future of young family members. Carers expressed that they felt a deep sense of sadness, grief and fear when thinking about the genetic implications of the disease on their children, and feeling a sense of helplessness in their ability to support their children in the future, as exemplified in the excerpt below. ## Adapting, managing symptoms and receiving carer support All carers in the study identified the importance of learning to adapt to the caring role, and trying to find ways of managing the challenging symptoms, particularly the behavioural symptoms of their spouse. Carers also expressed the value of receiving support to meet the specific needs of spousal carers of people with bvFTD. ## Appreciating, adapting and living in the present Eleven carers described the benefits of holding an adaptive and positive mind-set in coping with and adapting to the role of becoming a spousal carer. Carers expressed the usefulness of practising appreciation for the positive aspects of their life that were maintained despite the deteriorating illness of their spouse. Examples of ways that carers practised this positive perspective-taking included comparing their situation to those who "have it far more severely" (C10), practising gratitude (C8 below), and implementing transferable skills from other caring roles they had previously occupied. Carers also expressed the helpfulness of planning for the future while also trying to focus on "living day to day" (C12). ## C8: now i realise that possessions don't mean anything […] you know, nobody, nothing can give you what you want back, which is your partner's health. so there's nothing out there that you need. so you then become just happy in your surroundings with what you've got, and just living that life now and making it the best for them that you can, (female, bvftd). Carers of people with fFTD expressed a "bubble of hope" (C14) that came from participating in a trial treatment, which they believed might halt the deterioration of the illness for their spouse, or could offer hope for future generations. ## Managing challenging behavioural symptoms of ftd Behavioural symptoms described as challenging included lack of insight, apathy and compulsivity. These changes were described as confusing, sad, and difficult to manage in public or embarrassing at best. At their worst, they placed the person with bvFTD and others at increased risk of harm. Therefore, carers put in place strategies to manage challenging and risky behavioural symptoms. As C10 describes the experience of trying to manage these risks below: ## C10: he was going to hang himself in the woods in the park […] and then [when we came back home] i wondered why he was nearly falling over. i realised it's because he was picking up alcohol and drinking […] and yes, i had to empty our house of alcohol and knives and everything. (female, fftd) Other carers discussed applying other restrictions to reduce risk, which included withholding money to reduce the risk of being taken advantage of by others, or to reduce risk of compulsive spending (C3) or controlling access to food (C14, C4, C6). Other carers described a difficult experience of having to manage their spouses' risky behaviour toward others, including violence and aggression. ## C8: he started to dislike that son and then he started to dislike me. […] and then that was when the violence started. […] and i didn't let him in the house for a week. i just was so confused. i was so upset. […] he'd have this outburst and then a while later it was, we're all calm now […] and he never spoke about it. (female, bvftd) Another key difficulty for carers was managing their spouse's behavioural symptoms in public. Carers described needing to put a system of control in place, to monitor, supervise or limit their spouse's behaviour when in a public space as exemplified by C10 below. C10: Here was that feeling of embarrassment for me as well, because he comes out and says odd things. And I don't like that. I'm a very quiet, private person, and things like this I find extremely hard. Yes, that's a bit hard-more than hard to accept. (Female, fFTD) ## Information gathering and linking to specialist services All of the carers acknowledged the value of acquiring knowledge about FTD, connecting with available resources to provide information, advice and carer support or respite. Thirteen carers described the value of obtaining information to educate themselves about the specific symptoms of bvFTD to help them manage expectations of disease progression, and provide helpful tips and resources to support them in their caring responsibilities. Carers did this by seeking out opportunities to learn about FTD from online sources, professionals and other carers. Eleven carers described the helpfulness of speaking with family, friends, other carers and professionals to gain an understanding of what to expect, and to normalise challenges faced in caring for a person with FTD. Others (e.g. C11) discussed that specialist FTD support was "much more helpful" than generic support, highlighting the necessity for services to accommodate for the specific needs of carers of people with FTD. These were understood to be different from those caring for people with more general forms of dementia. Six carers identified the importance and usefulness of accessing information and emotional support online. Three participants expressed mixed feelings about the value of connecting with fellow FTD carers whilst acknowledging that there may be a time in the future when they would find attending carer support groups helpful. Whilst not all carers agreed on the value of using carer support groups, 13 carers identified carer respite as one of the most essential needs amongst spousal carers of people with FTD. ## C1: i realised that being together 24/7 was a nightmare for both of us, and that i needed a break, but he needed a break away from me. […] i decided that i really wanted to go to [dementia charity choir] so i got a carer […] i go back to choir once a week, and a girl comes in to sit with him, Other carers described similar experiences of attending social activities run by dementia support charities, and described them as helpful in enabling them to continue to participate in activities and hobbies of interest while receiving support and respite from daily spousal caring. These connective spaces for carers provided an opportunity to spend time with others with a shared understanding of the carer's experience. For many, this formed an important part of the carer's social supportive network post-diagnosis. ## C10: i am grateful that i have got a lot of friends and people i knew, and the vast majority coming in now are people we know from the dementia cafes and charity things. (female, fftd). Outside of informal social support and networking spaces, 10 carers highlighted the importance of attending medical follow-ups or receiving nursing support as a way of providing on-going support after diagnosis and during the care-recipients' lifespan. ## C8: i've used admiral nurses [registered nurses who specialise in dementia]. i've used those quite a few times and they are lovely. they are so supportive and so understanding, because he's been very, very violent to my neighbours (female, sporadic bvftd). Five of the nine participants caring for a spouse with fFTD expressed the value of having prior knowledge about dementia from their spouse's family. This knowledge, prior to diagnosis, was helpful in preparing them to manage expectations. ## C1: we've known for quite a long time, because his sister had it…, (female, fftd) The excerpts above highlight the needs of carers by identifying the ways in which they adapt to the new carer role, manage symptoms, and receive crucial support from healthcare services, specialist dementia support and FTD carer support spaces. ## Lack of general knowledge-barriers to support However, despite carers being able to acknowledge key support that they found helpful, carers also identified significant barriers to accessing and receiving support necessary to help them in their roles as carers of a spouse with FTD. Twelve carers identified that the most significant barrier to receiving support was a lack of general knowledge about FTD, particularly amongst professional support services. ## Lack of appropriate services available For 12 carers this lack of awareness about FTD meant that carers often felt that there were limited resources suitable to meet their specific needs. ## C14: and with the support group for dementia, [husband] will walk in and he'll go, "yeah, i don't look anything like these people. (female, sporadic bvftd) Carers commonly described the younger age of their partner and the unique presentation of FTD behavioural symptoms as barriers to receiving appropriate support, C4: One of the problems I had when looking for respite for was that so many of the homes, you actually look and they all say, Oh yeah, we take people from the age of 65"[…] the thought of him at 55, being in a home where the next youngest person is 70. For carers, advice and support received from more general dementia support often did not seem appropriate to meet their specific carer needs. C6 describes how she felt different, and unable to relate to others when attending a dementia carer support course. ## C6: i'm doing a course through the alzheimer's society […] there's no one else young and there's no one else with ftd. and it is becoming apparent over the weeks how different my situation is to their situation. (female, fftd) ## Limited accessibility and provision of services Eight carers described having knowledge of an available service, but with barriers that interfered with their ability to access valuable services. Barriers included carers' lack of knowledge about available resources and limited available resources in their area. ## C4: yeah, the thing is, i find support quite confusing […] i get to the stage where i ask, "well who are the people i see for this, and that?" […] i am aware that there is a reasonable amount of support in [my area], but as i say, it's just knowing who to contact. (female, sporadic bvftd) Carers experienced a general lack of awareness about FTD amongst healthcare services, which may be linked to their experience of having limited availability of specialist support to meet the unique needs of spousal carers of people with bvFTD. # Discussion This is the first study to explore the experiences of spousal carers of people with bvFTD identifying support needs and barriers to meeting those needs. The findings highlight complex processes involved in receiving a diagnosis and adjusting to the role of spousal carer. This was impacted by the subtle onset of the condition, and further complicated by the challenging route to diagnosis and continued lack of support following diagnosis. Adjusting to the life altering, relationship changing and family impacts of becoming a carer to a person that is also a spouse was crucial in defining the spousal carer experience of bvFTD. In line with previous literature [bib_ref] Frontotemporal Dementia: An Updated Clinicians Guide, Rosness [/bib_ref] over half the carers experienced delays in diagnosis often because gradual behavioural symptoms were misinterpreted. The current study found that the lack of knowledge about FTD had a negative impact on carers. Information about common forms of dementia was insufficient due to the unique behavioural changes seen in bvFTD, which have a significant impact on spousal relationships. This lack of knowledge amongst healthcare professionals and the general public contributed to increased feelings of isolation, confusion, distress and frustration by carers. This meant that carers needed to play an active role in reaching diagnosis and advocating for the care of their spouse. Extending previous literature, carers described the complex process involved in receiving the bvFTD diagnosis, including experiencing mixed emotions, of relief, fear and a continued sense of isolation. There is a significant emotional impact of pre and post-diagnostic stages on carers which echoes findings from Chow and colleagues [bib_ref] An international needs assessment of caregivers for frontotemporal dementia, Chow [/bib_ref]. This is a time when carers are likely to be in greatest need of information on the symptoms of FTD [bib_ref] Experiences and needs of spouses of persons with young-onset frontotemporal lobe dementia..., Johannessen [/bib_ref]. Findings support existing literature emphasising that the receipt of diagnosis encompasses a difficult shift in the spousal relationship, which continues throughout the progression of FTD, and is characterised by a loss of communication, deep emotional connection, and companionship [bib_ref] Family caregivers experiences of the pre-diagnostic stage in frontotemporal dementia, Rasmussen [/bib_ref]. These changes are affected by behavioural symptoms of lack of empathy, apathy, loss of motivation and disinhibition, which often act to distance spouses or make communication and mutual understanding difficult [bib_ref] Family caregivers of individuals with frototemporal dementia, Wong [/bib_ref] , and therefore likely describe an experience that is specific to spousal carers of people with bvFTD. Carers likened this experience to having lost a partner that is still living with them. Previous qualitative studies describe how carers feel deep feelings of sadness and grief [bib_ref] Relatives' experiences of frontal-variant frontotemporal dementia, Oyebode [/bib_ref] as the spousal relationships becomes replaced by that of carer and care-recipient [bib_ref] Caring for loved ones with frontotemporal degeneration: The lived experiences of spouses, Massimo [/bib_ref]. Furthermore participants described feelings of confusion and fear in response to the unpredictable nature of the behavioural changes, which at times included aggression, violence and high levels of risk, suggesting specific challenges and a greater emotional toll on carers of people with bvFTD, compared to other dementias. This is in line with prior studies describing the unique challenges of the FTD caring experience [bib_ref] An international needs assessment of caregivers for frontotemporal dementia, Chow [/bib_ref] and a greater burden [bib_ref] Experiences and needs of spouses of persons with young-onset frontotemporal lobe dementia..., Johannessen [/bib_ref] and distress [bib_ref] Impact of behavioural problems on spousal caregivers: A comparison between Alzheimer's disease..., Vugt [/bib_ref] in caregivers of people with FTD compared to those with Alzheimer's disease. Furthermore, behavioural changes have also been associated with caregiver depression in those caring for people with FTD [bib_ref] Patient and Relative Experiences and Decision-making About Genetic Testing and Counseling for..., Crook [/bib_ref]. Not previously reported in FTD carer literature this study explored the unique experiences of spousal carers of individuals with fFTD. This addressed the important implications of genetic disease on the family and included, issues surrounding testing and disclosure, parental feelings of helplessness regarding their ability to protect younger generations from developing FTD, and fearing for their family's future. Although carers described mixed feelings about testing and disclosure, they acknowledged that knowing about the condition prior to diagnosis was helpful in preparing to manage expectations of their future together, and facilitated a process of accepting the diagnosis and adjusting to the carer role. A systematic review by [bib_ref] Patient and Relative Experiences and Decision-making About Genetic Testing and Counseling for..., Crook [/bib_ref] found that while no studies specifically assessed the experience of genetic testing in fFTD, the wider FTD literature suggests that the carers and relatives of those with FTD often carry the responsibilities of providing consent on behalf of the patient and notifying family of the test results [bib_ref] Patient and Relative Experiences and Decision-making About Genetic Testing and Counseling for..., Crook [/bib_ref]. Carers developed ways of coping, some of which related to learning and implementing specific behavioural strategies, taking time for respite or shifting attitudes and perspectives towards their circumstances [bib_ref] Caregiver burden, healthrelated quality of life and coping in dementia caregivers: A..., Riedijk [/bib_ref]. For example, participants described using strategies to manage challenging and risky behavioural symptoms, and supervising behaviour in a public, in line with reports from previous qualitative research [bib_ref] Relatives' experiences of frontal-variant frontotemporal dementia, Oyebode [/bib_ref]. Due to the progressive and changing presentation of the disease, carers' coping must be adaptive. Therefore, carers' responses indicate that the ability to cope fluctuates over time and this may be applied to the framework of the Lazarus and Folkman's transactional model, which states that coping is not fixed and is dependent on an evolving relationship between an individual and their environment, as well as an individual's appraisal of a stressor and their perceived ability to cope. As the disease progresses, caregiver's appraisal of their ability to cope may decrease, resulting in an increased stress response. The most important barrier reported by carers was the general lack of awareness about FTD, which impacts on diagnostic delays and support offered to carers. Participants expressed that they did not "fit in" amongst generic dementia support services, as they were insufficient in meeting the specific needs of spousal carers of people with bvFTD. Often due to younger age, familial and genetic implications and unique presentation of symptoms which dramatically impact on the spousal relationship and require specific behavioural and psychosocial coping skills on the part of the spousal carer. Therefore, findings emphasise a crucial need to educate healthcare professionals about FTD [bib_ref] Family caregivers of patients with frontotemporal dementia: An integrative review, Caceres [/bib_ref] , and the initial symptoms and presentations, so that carers may be able to address concerns, receive timely diagnosis and access appropriate support [bib_ref] Family caregivers experiences of the pre-diagnostic stage in frontotemporal dementia, Rasmussen [/bib_ref]. ## Limitations and future directions The current study included more participants than other qualitative studies in the area of carer experiences of FTD [bib_ref] Caring for loved ones with frontotemporal degeneration: The lived experiences of spouses, Massimo [/bib_ref] [bib_ref] Relatives' experiences of frontal-variant frontotemporal dementia, Oyebode [/bib_ref] , and is an appropriate sample size for the TA. However, all participants were White British, residing in UK geographic areas comprising a majority of residents from upper income households and mostly female, which should be considered when addressing the transferability of the findings. Similarly, the sample also comprises of participants who are part of large cohort studies in a specialist dementia centre, therefore they are likely to be engaged with support and research teams and as such their experiences may not be generalisable to the wider carer experience. In addition, the reluctance of participants to personally identify as "carers" may hold implications that could provide information regarding crucial processes involved in transitioning into the carer role and the meaning of identifying as a carer or spousal carer. These were not explicitly taken into account during the process of the thematic analysis, which could have omitted this key aspect of the participants' experience. Furthermore, while the researcher attempted to remove implicit biases, this heuristic frame of the "carer and patient", as opposed to the "participant and spouse" may have biased the interpretations influencing the double hermeneutic process [bib_ref] Interpretative phenomenological analysis, Smith [/bib_ref] ,whereby the researcher was attempting to make sense of the participant (as a carer) making sense of their experience (as a spouse and not a carer). # Future research The current study did not explore the impact on children living in the home during the early years of the disease, which may be of particular interest given that many carers report noticing symptoms up to twelve years before receiving a diagnosis, and acknowledge challenges in managing the responsibilities that come with a young family, alongside increasing responsibilities of caring. It will be important for research to further investigate the complex needs of young families with bvFTD, as well as explore the implications of the genetic condition on the family system. ## Clinical implications Guidance by the National Institute of Health and Care Excellencerecommends that psychosocial support be provided for carers of people with dementia. However, the present study found that generalised dementia support offered to carers of people with FTD are insufficient to meet their needs. Therefore, it is important to emphasise public awareness campaigns and health professional education on bvFTD, as well as offering appropriate ongoing support [bib_ref] Family caregivers experiences of the pre-diagnostic stage in frontotemporal dementia, Rasmussen [/bib_ref]. For example in the UK, educating general practitioners to improve the pre-diagnostic stage and journey to diagnosis and providing on-going support through memory services may be beneficial. Furthermore, families of people with fFTD may further benefit from receiving additional support regarding the genetic implications of the disease. Psychosocial interventions should be offered to support patients and families to cope with the specific challenges of bvFTD. Psychosocial interventions should include managing challenging behavioural symptoms, learning adaptive psychological coping strategies, engaging in FTD carer support groups as well as psychosocial support relating to grief, loss and processing the relationship change [bib_ref] Impact of behavioural problems on spousal caregivers: A comparison between Alzheimer's disease..., Vugt [/bib_ref] [bib_ref] Specific support needs and experiences of carers of people with frontotemporal dementia:..., Tookey [/bib_ref] [bib_ref] Caregiver burden, sleep quality, depression, and anxiety in dementia caregivers: a comparison..., Liu [/bib_ref] [bib_ref] The social and economic burden of frontotemporal degeneration, Galvin [/bib_ref] [bib_ref] Caregiver Burden and Needs in Frontotemporal Dementia, Diehl-Schmid [/bib_ref]. # Conclusion Healthcare professionals should be educated on the initial symptoms and presentations of FTD, so carers can address concerns, receive timely diagnosis and appropriate support. Families of people with FTD may benefit from receiving additional support in managing the genetic implications of the disease. Future research should investigate the needs of carers and families or people with fFTD, and would benefit from investigating positive or meaningful experiences in caring which may inform development of adaptive coping processes. [fig] 1: Participant Demographics Key: FTLD-CDRSOB (O'Bryant et al., 2008): 4-7.5 (lowest severity); 8-.5 (moderate severity); 2-5.5 (high severity); >6 (severe) Multiple deprivation Indices (Dept. for Communities & Local Government, 209): is most deprived; 0 is least deprived Diagnosis: fvFTD = bvFTD with familial; bvFTD = bvFTD only [/fig] [table] C7: He was diagnosed with bipolar, hypomanic, bipolar or something. […] See I'd Googled, as you do, and I thought he'd got Pick's disease. And I told the people at the memory clinic, "I think it's this." And they said, "It's not. He's passed his memory test." (Female, fFTD) [/table] [table] C3: If you think about it, you're managing somebody's life who's there with you, but you can't really have any discussion about it what-so-ever, because if you do, it's forgotten about in seconds and he doesn't understand the implications. [...] He doesn't have that emotional response really. [/table]

Complementary feeding practices, dietary diversity, and nutrient composition of complementary foods of children 6–24 months old in Jimma Zone, Southwest Ethiopia Background: Mothers and caregivers typically feed infants according to their culture, purchase power and level of awareness with no due diligence to nutritional quality of the diet. Scientific evidence on nutritional adequacy of predominant complementary foods is critical for planning and prioritising interventions. The purpose of the current study was to evaluate the quality of complementary foods and the optimality of complementary feeding practices in Southwest Ethiopia. Methods: In this cross-sectional study, a stratified multistage sampling procedure was used to sample 433 children, 6-24 months old. A semi-structured questionnaire was used to collect demographic, socio-economic and dietary data. Dietary diversity score was measured using a 24-h dietary recall. Six customary complementary food types were assayed for proximate composition, energy and mineral density using standard methods. Adequacy of the complementary foods in nutrients for complementary feeding purposes was assessed as a ratio between actual composition and recommended composition of complementary foods. Results: Only 16.1% of the children get the minimum dietary diversity. The children were reported to be fed with cereals & grains (68.8%), discretionary calories (53.6%), protein-rich foods (44.6%), oils and fat (40.5%), vegetables (38. 5%), dairy products (17.9%) and fruits (28.1%). The sampled foods contained 4.3-24.4%, 0.9-8.5%,mg/100 g, 1.8-4.1 mg/100 g and 22.5-42.4 mg/100 g of total carbohydrate, crude fat, protein, energy content, calcium, zinc and iron, respectively. All the complementary food samples predominantly fed to children were not composed of adequate protein, fat, carbohydrate, energy and calcium as recommended for complementary feeding purposes. However, most of the complementary foods are composed of adequate iron and zinc. Conclusions: The nutrient density and diversity of complementary foods of 6-24-month-old children in the study area were found to be sub-optimal. Upgrading the nutritional composition of the starchy complementary foods should be of highest priority to improve nutrition of the infants and young children. # Background Complementary feeding refers to supplementing breastfeeding with feeding children aged between 6 and 24 months with a wide range of foods. The period between 6 and 24 months of age is a time of nutritional vulnerability because during this period, nutrients especially micronutrients and energy obtained only from breast milk will not be sufficient to meet the requirements of the child. Ensuring adequate nutrition during the period between 6 and 24 months of age is a major global health priority. Among the immediate causes of undernutrition among children is consumption of too few nutrients. In most low-income countries, including Ethiopia, the beginning of growth faltering coincides with the start of complementary feeding; age-specific malnutrition rates generally increase until about 24 months of age and then level off. The sharp rise in the occurrence of stunting in young children from the age of 6 months is usually associated with suboptimal complementary feeding practices. As children younger than 24 months old do not consume a sufficient amount of food to cover the high nutrient needs for growth and development, food given to them should be of high nutrient density. Theoretically, infants should receive the most nutrient-dense diet in the family. Infants in low-income countries, however, are typically fed with nutrient-poor foods like thin porridges. Complementary foods should contain high-biological value protein, furthermore, vitamins and minerals. The National Nutrition Strategy (NNS) of Ethiopia gives considerable emphasis for nutrition of children younger than 2 years old in particular as nutrition received during this period influences how the children develop, grow and learn now or later. There is no documented evidence of overall complementary feeding practices and adequacy of the complementary foods in nutrients in Jimma Zone. This information is critically needed to be able to judge and plan the mechanisms to upgrade traditional diets. Therefore, this study aimed to evaluate the complementary feeding practices, dietary diversity and nutrient adequacy of complementary foods of children 6-24 months old in Jimma Zone, Southwest Ethiopia. # Methods ## Area and subjects This study was conducted in Jimma Zone, Southwest Ethiopia. The study area is year-round green but unfortunately characterised by household food insecurity. Three districts were purposively selected based on their agricultural production; Omo Nada, Dedo and Mana are cereal, vegetable and cash crop producer areas, respectively. This study is a component of a more prominent cross-sectional study which assessed the nutritional status and associated factors among children younger than 2 years old. The study population for the original research included all children younger than 2 years old in the study area. A multistage stratified sampling procedure was used to sample 558 children who were 0-24 months old. For the current study, only those children 6-24 months old were included. ## Data collection Data were collected from mothers or caregivers of the infants and children using face-to-face interviews using a semi-structured questionnaire. ## Variables The variables were categorised as dependent and independent variables. The dependent variable was the dietary diversity score of the 6-24-month-old children. The independent variables included several socio-economic and demographic factors like family composition, household size, educational level attained by mothers and fathers, the occupation of mothers and fathers, the wealth of the household and education or training received on health and nutrition. Additionally, the infant-and young child-feeding (IYCF) practices were also assessed. ## Measurements ## Diet diversity A single 24-h dietary recall was used to obtain data on dietary diversity. Dietary diversity was assessed with a scale of seven food groups namely cereals and grains, vegetables, fruits, dairy products, oil and fat, protein-rich foods and discretionary calorie foods. Dietary diversity score (DDS) was found to be optimal when a child is fed greater than four food groups per day. ## Nutrient composition of complementary foods First, the complementary foods fed to 6-24-month-old children were identified from the questionnaires. Complementary food samples (25 g) were collected from 217 households. The collected food samples were labelled and stored at − 18°C and eventually transferred to Jimma on the same date of collection. Based on the ingredients used for making the complementary foods, 6 assays were identified. The gross sample was then reduced in size and homogenised to create the laboratory sample. The samples were then analysed for proximate composition (protein, fat, carbohydrate, moisture, ash and fibre), energy content, mineral (iron, zinc, calcium and phosphorous) and anti-nutritional factors (phytate and tannin) following the respective standard methods of analysis. ## Nutrient adequacy of the complementary foods Adequacy of the complementary foods in nutrients for complementary feeding purposes was assessed as a ratio between actual composition and recommended composition of complementary foods. # Statistical analysis The data were analysed using Statistical Package for Social Sciences software version 20 (SPSS Inc., Chicago, IL, USA). Descriptive statistics such as percentages were calculated. Bivariate analysis was conducted for the dietary diversity data. P values of less than 0.05 were regarded as statistically significant.presents the socio-demographic characteristics of the respondents. Fifty four percent of the children in this study were male and 46% were female. Four hundred thirty-three out of 558 children included in the study were aged 6-24 months old. The larger part of the participants 372 (66.7%) were rural residents. A majority of the participants 516 (92.5%) was Muslims in religion. More than half of the children 306 (54.8%) were third and above in their birth order. Additionally, majority of the mothers 360 (64.5%) were aged 20-29 years old. Regarding literacy, larger part of the participants 336 (60.2%) did not attend formal education. A large proportion of the mothers 436 (78.1%) were housewives. # Results ## Characteristics of the sample Complementary feeding practicesshows the infant-and young child-feeding practices in the study area. The majority (88.9%) of the children were exclusively breastfed, and 75.6% were breastfed up to the age of 2 years. Both early and late initiation of additional food was practised extensively in the study area, but most (82.9%) of the mothers started to give complementary food to their children just at 6 months. However, nearly half of the mothers (53.8%) do not prepare any particular complementary food other than the typical family dish while the rest make some other additional foods of which gruel or Atmit is the predominant one. At the same point, almost all (91.6%) of the mothers do not prepare any particular food to their children during sickness or recovery from disease. The study also signified that 96.7% of the mothers feed their children 3-4 times a day. Regarding dietary intake, two thirds (68.8%) of the study participants consumed cereal-based gruel (made of barley, oat, teff, wheat, sorghum). Nearly half (44.6%) of the study participants reported that they fed their children with protein-rich food before the survey and (53.6%) of the study subjects consumed discretionary calories in the previous 24 h. Fruits, vegetables and dairy products were consumed by 28.1%, 38.5% and 17.9% of the participants, respectively.presents the dietary diversity of the study participants. Majority of the children (83.9%) did not get the minimum dietary diversity. Dietary diversity score was significantly (P < 0.05) influenced by the age and birth order of the child, maternal and paternal education, socio-economic status of the family and paternal occupation. Higher dietary diversity was scored among children aged 12-24 months old, whose birth order was first and whose parents were formally educated. Children whose fathers are not farmers and children living in homes with higher wealth status also had higher DDS. ## Dietary diversity ## Nutrient composition of complementary foods The macronutrient composition and energy contents of the complementary foods are presented in On the other hand,presents the micronutrient and anti-nutrient composition of complementary foods dominantly consumed by the children. The mineral content ranged between 168.41 and 250.4 mg/100 g, 1.78 and 4.14 mg/100 g, 22.48 and 42.39 mg/100 g and 225.56 and 317 mg/100 g for calcium, zinc, iron, and phosphorus, respectively. The anti-nutritional factor contents ranged between BDL (below detection limit) of 117.72 mg/100 g for phytate and 1.17-75.17 mg/100 g for tannin. Nutrient adequacy of complementary foods for complementary feeding purposespresents nutrients adequacy of the studied complementary foods for complementary feeding purposes. All of the foods did not contain adequate amounts of protein, fat, energy, zinc and calcium (values < 1. On the other hand, most of the diets provide sufficient quantities of ash and iron (values > 1). The requirements were not met for any of the nutrients. # Discussion ## Complementary feeding practices Studies in developing countries showed that both too early and a too late introduction of complementary food is familiar. Mothers in South Africa start complementary feeding within 2-3 months. In Uganda, 44.1% and 27% mothers began complementary feeding within 2-3 months in 1997 and 2005, respectively. Our findings oppose these reports because for the majority of the children in the study area complementary feeding is initiated just at the appropriate time. Contrary to the recommended practice of complementary feeding, the majority of the mothers do not prepare particular food during sickness or recovery. During an illness, the need for fluid often increases, so a child should be offered and encouraged to take more. However, feeding frequency in the study area was optimal for children in the age group of 6-12 but not for those in the age group of 13-24 months. The recommended number of meals per day for a healthy breastfed baby should be 2-3 times at 6-8 months, 3-4 times at 9-11 months and 3-4 times with 1-2 additional nutritious snacks at 12-23 months of age. The predominant complementary food fed to children in the study areas is gruel (a liquid drink made of cereals), locally named Atmit, ranking first in the all of the three districts. In Ethiopia, 70% of children aged 6-23 months, predominantly consume foods made of grains. Similarly, a study conducted in Nigeria reported that the dominant food groups in the children's diet were cereal/grains. Nutritional problems are common among populations whose diets are predominantly based on starchy staples, and these plant-based foods are low in micronutrient contents, high in phytate and high in dietary fibre which inhibits the absorption of micronutrients. CODEX CAC/GL 08Dietary diversity The results of the current study showed that nearly half of the children had sub-optimal dietary diversity. A survey of community-based production complementary foods in four regions of Ethiopia has reported that the highest mean diversity score of 3.4 was observed in Oromia region, followed by Tigray (2.9) Amhara (2.7) and SNNP (2.6) regions. In Oromia region, nearly half (49.4%) of the children had a suboptimal diet diversity. According to our results, children from wealthier households consumed more diversified diets. The ability to produce sufficient food for one's household at home and generate enough revenue to purchase foods on the market are ways that a family could achieve food security. The results of this study also indicated that girls are fed with less diversified diet compared to their male counterparts. Male gender bias in the intra-household distribution of food and other resources has been reported from Ethiopiaindicating that girls are less favoured in the resource-constrained environments. ## Composition of diets The complementary foods were predominantly made of starch-based cereals and hence of poor nutritional value, and do not satisfy the infant's basic needs of protein because they have limited levels of protein both qualitatively and quantitatively. As well, macro-and micronutrients may be insufficient to maintain growth and development, this results in reduced nutritional status in children. ## Nutrient adequacy of the complementary foods for complementary feeding purposes Complementary foods should contain the following amount of estimated macronutrients: moisture content of ≤ 5%, an ash content of ≤ 3%, a crude protein content of ≥ 15%, a crude fat content of 10-25%, a crude fibre content of ≤ 5% and a carbohydrate content of 64 ± 4%. Additionally, the energy content of 400-425 kcal/100 g, a calcium content of 500 mg/100 g, the iron content of 16 mg/100 g and zinc content of 3.2 mg/100 g are also required. Children younger than 2 years old and living in developing countries should consume approximately 137-187 g/day, 206-281 g/day and 378-515 g/ day of complementary foods at the age of 6-8 months, 9-11 months and 12-24 months, respectively to meet their energy needs. The total nutrient and energy requirements of healthy breastfeeding infants have been established. The average nutrient and energy that a given complementary food should provide are estimated by subtracting average nutrient and energy content of breast milk from the total nutrient and energy requirement at each age. The complementary food samples were not adequate in nutrients for complementary feeding purposes. Over and under-reporting of infant dietary intakes is among the reasons that made quantifying infants' diets difficult. Parents tend to over-report intakes for they do not wish to be seen either to be under-feeding babies or on the one hand not being able to distinguish between food offered to young children and the amount consumed. # Conclusions The feeding practices of 6-24-month-old children in the study area were not satisfactory. Dietary diversity and macronutrient, energy and overall nutrient composition of the complementary foods were below the recommendations. The complementary foods were found to contain adequate amounts of iron. Improving the nutrient adequacy of the locally available customary diets through food processing techniques and a community-based nutritional education on optimal child feeding are recommended. ## Availability of data and materials The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. Authors' contributions SFF, TB and OH conceived and designed the experiments. NK and SFF collected the data. SFF, NK and TB analysed the data. SFF, TB and OH contributed reagents/materials/analysis tools. SFF prepared the draft manuscript. TB and OH finalised the manuscript and provided a critical review. All authors have read and approved the final manuscript. Ethics approval and consent to participate Ethical clearance was obtained from the Research and Ethical Review Board of Jimma University. Permission to undertake the study was obtained from every relevant authority in Jimma Zone. Each study participant was briefed about the study and offered the opportunity to ask questions. Then oral informed consent was obtained from each participant prior to participation in the study and data was kept confidential. The Ethical Review Board

Pregnancy through Assisted Reproductive Technology in a Patient with Thoracic Endometriosis Syndrome [fig_ref] Figure 2: Computed tomography chest [/fig_ref] [bib_ref] Bronchobiliary fistula: A rare complication of hepatic endometriosis, Schuld [/bib_ref] [bib_ref] Synchronous rectovaginal, urinary bladder, and pulmonary endometriosis, Hilaris [/bib_ref] [bib_ref] Catamenial pneumothorax: A rare entity? Report of 5 cases and review of..., Visouli [/bib_ref] [bib_ref] Abdominal wall endometrioma mimicking an incarcerated hernia: A case report, Simoglou [/bib_ref] [bib_ref] Endometriosis of the lung: Report of a case and literature review, Huang [/bib_ref] ## Case report A 29-year-old female married for 4 years, presented with subfertility, with no menstrual complaints. All the basic investigations of the couple were normal, including folliculometry. She never gave a history suggestive of endometriosis or other pelvic pathology. Hence, she was advised for a diagnostic hysterolaparoscopy in November 2014. During the routine preoperative workup, an X-ray chest done in the immediate postmenstrual phase showed right-sided hydropneumothorax [ [fig_ref] Figure 1: Right hydropneumothorax Journal of Human Reproductive Sciences ¦ Volume 11 ¦ Issue... [/fig_ref] ]. Hence, the surgery was postponed and reference revealed the same findings, and hence, an intracostal drainage was inserted []. When the drain was in situ, the patient had menstruation with complaints of chest pain in the right side of the chest and the drain tube had more blood, which clinched the diagnosis of "catamenial pneumothorax." The patient was subjected to video-assisted thoracoscopy (VATS) with bronchoscopy on January 28, 2015, in which ectopic endometrial tissues were seen over pleura, lung, as well as parts of diaphragm, were subjected to biopsy and sent for histopathology and culture, and were cauterized. Pneumatocele was identified on the right lobe, for which staples were applied. The histopathology confirmed pleural endometriosis. The tissue was subjected to immunohistochemistry which confirmed the tissue to be positive for estrogen receptor, progesterone receptor, CD-10 -consistent with pleural endometriosis [ [fig_ref] Figure 4: Histopathology report [/fig_ref] ]. As per the patient's desire, she was subjected to pleurodesis with steritalc and was put on injection leuprolide for 6 months. This gave her good relief from right-sided chest pain with amenorrhea for 9 months. The patient was monitored with a series of chest X-rays and CT scans. Serum CA-125 was not performed in this patient. The patient returned for infertility treatment in April 2016. Hysteroscopy with uterine septum resection (1-1.5 cm fundal septum -cut with scissors) was carried out on May 26, 2016. Laparoscopy was avoided as the patient had persistent right pleural effusion [ [fig_ref] Figure 6: Persisting right pleural effusion [/fig_ref] ] (decreased than before). After detailed counseling, including all options, the couple wished to undergo self-stimulation and transfer. Patient's anti-Müllerian hormone was 1.65. The patient was started on IVF antagonist protocol in July 2016 with recombinant follicle-stimulating hormone 225 i.u. daily from day 2 of menstrual cycle. Ovum pick up was done on July 25, 2016, and seven oocytes were retrieved. Six embryos obtained on day 3 (3-8 cell A, 2-8 cell B, 1-8 cell C) and the embryos were electively cryopreserved. The patient received leuprolide depot for one cycle, and frozen embryo was planned in the next month with estradiol valerate 6 mg/day, from the 3 rd day of cycle. After 12 days of estradiol valerate, once the endometrium was satisfactory, progesterone support was given and three embryos (2 Grade A, 1 Gr B) were transferred on August 24, 2016. The patient conceived in the same cycle and was followed up closely throughout the 9 months of pregnancy, which was uneventful. At 36 weeks of gestation, the patient presented with acute urinary retention and elective cesarean section was done on April 19, 2017, and a male child weighing 2.4 kg was delivered. Uterine surface had endometriotic blebs with increased vascularity [ ]. The patient was discharged on day 6 of delivery. Lactation was also well established and has been encouraged to continue breastfeeding till at least 1-year postpartum. An X-ray chest was taken 3 weeks postpartum [ [fig_ref] Figure 8: X-ray chest postoperative Figure 7 [/fig_ref] ]. # Discussion TES is a challenging clinical entity. A high index of clinical suspicion is of paramount importance as both diagnosis and treatment may often be delayed for years. The present case had an important feature that the patient never gave any history suggestive of pelvic The detection of catamenial pneumothorax with the intercostal drainage during her menstrual cycle led to the diagnosis of pulmonary endometriosis, which in this patient was mostly only pleural and on the right side. Literature review does suggest that pleural endometriosis is more frequent on the right side, [bib_ref] Catamenial pneumothorax revisited: Clinical approach and systematic review of the literature, Korom [/bib_ref] as seen in this patient. Most of the cases of pleural endometriosis are associated with severe pelvic endometriosis [bib_ref] Thoracic endometriosis syndrome: New observations from an analysis of 110 cases, Joseph [/bib_ref] though this patient never complained about it, and the presence was detected only at the time C-section. The literature is still inconclusive about the best treatment strategy in the presence of TES in infertile young patients. Pleurodesis, especially in younger patients, should always be carried out concomitantly with VATS exploration. This way, an accurate diagnosis and treatment can be instituted and recurrences avoided. In older women and those not desirous of pregnancy, hysterectomy and bilateral salpingo-oophorectomy (BSO) is the treatment of last resort when other options have failed. Hence, a multidisciplinary approach by thoracic surgeon and gynecologist carries the highest chance of making an accurate diagnosis and providing the appropriate treatment strategies, [bib_ref] Endometriosis of the diaphragm: Four cases treated with a combination of laparoscopy..., Nezhat [/bib_ref] as seen in this case. Retrospectively, the decision to plan IVF-ET was the right choice for this patient and interestingly during the stimulation for IVF and subsequent transfer, and 9 months of pregnancy, the patient did not have any deterioration of the disease. ## Literature review To date, data are limited on the association between pelvic endometriosis and fertility status in patients with TES. In a recent series (2017) of 16 patients of TES by Ottolina et al., [bib_ref] Thoracic endometriosis syndrome: Association with pelvic endometriosis and fertility status, Ottolina [/bib_ref] the study supported the presence of a strong association between catamenial pneumothorax and pelvic endometriosis. Pelvic endometriosis was diagnosed in 9 (56.3%) of the patients with TES. However, six patients did not undergo laparoscopic procedure to confirm or exclude the disease. Seven of the affected patients (77.8%) had moderate-to-severe disease (Stage III-IV), four conceived spontaneously after laparoscopic surgery, and all patients who did not have pelvic endometriosis conceived spontaneously. Although the study concludes that women affected by TES should be reassured about their childbearing capacity, a large study should be undertaken to definitely clarify this aspect. ## Declaration of patient consent The authors certify that they have obtained all appropriate patient consent forms. In the form, the patient has given her consent for her images and other clinical information to be reported in the journal. The patient understands that name and initial will not be published and due efforts will be made to conceal identity, but anonymity cannot be guaranteed. ## Financial support and sponsorship Nil. ## Conflicts of interest There are no conflicts of interest. [fig] Figure 2: Computed tomography chest [/fig] [fig] Figure 1: Right hydropneumothorax Journal of Human Reproductive Sciences ¦ Volume 11 ¦ Issue 2 ¦ April-June 2018 Krishnakumar, et al.: Pregnancy through ART in a patient with TES endometriosis, such as dysmenorrhea and dyspareunia. [/fig] [fig] Figure 4: Histopathology report [/fig] [fig] Figure 6: Persisting right pleural effusion [/fig] [fig] Figure 5: Immunohistochemical report [/fig] [fig] Figure 3: (a) International Classification of Diseases -lateral view. (b) International Classification of Diseases b a [/fig] [fig] Figure 8: X-ray chest postoperative Figure 7: Intraoperative findings [/fig]

Targeted metagenomics as a tool to tap into marine natural product diversity for the discovery and production of drug candidates Microbial natural products exhibit immense structural diversity and complexity and have captured the attention of researchers for several decades. They have been explored for a wide spectrum of applications, most noteworthy being their prominent role in medicine, and their versatility expands to application as drugs for many diseases. Accessing unexplored environments harboring unique microorganisms is expected to yield novel bioactive metabolites with distinguishing functionalities, which can be supplied to the starved pharmaceutical market. For this purpose the oceans have turned out to be an attractive and productive field. Owing to the enormous biodiversity of marine microorganisms, as well as the growing evidence that many metabolites previously isolated from marine invertebrates and algae are actually produced by their associated bacteria, the interest in marine microorganisms has intensified. Since the majority of the microorganisms are uncultured, metagenomic tools are required to exploit the untapped biochemistry. However, after years of employing metagenomics for marine drug discovery, new drugs are vastly under-represented. While a plethora of natural product biosynthetic genes and clusters are reported, only a minor number of potential therapeutic compounds have resulted through functional metagenomic screening. This review explores specific obstacles that have led to the low success rate. In addition to the typical problems encountered with traditional functional metagenomic-based screens for novel biocatalysts, there are enormous limitations which are particular to drug-like metabolites. We also present how targeted and function-guided strategies, employing modern, and multi-disciplinary approaches have yielded some of the most exciting discoveries attributed to uncultured marine bacteria. These discoveries set the stage for progressing the production of drug candidates from uncultured bacteria for pre-clinical and clinical development. ## Marine microorganisms as a novel source of natural products Natural products remain a major resource for drug production today and during the past 30 years, 70% of antimicrobials and 60% of chemotherapeutics have been developed or analogously synthesized from them [bib_ref] The oceans and human health: the discovery and development of marine-derived drugs, Pomponi [/bib_ref] [bib_ref] Diversity in natural product families is governed by more than enzyme promiscuity..., Grüschow [/bib_ref]. Traditionally, terrestrial sources have provided the bulk of natural products for drug molecules. However, participation by the major pharmaceutical companies declined in the mid-nineties, largely owing to the high rediscovery rate and decreased number of novel compound identifications [bib_ref] Drug development from marine natural products, Molinski [/bib_ref]. In the meantime infectious diseases and multiple drug resistant strains have bloomed, urging scientists to mine for novel drugs in nonterrestrial and unexplored environments. A chemoinformatics study showed that 71% of the marine natural products were not represented in terrestrial natural products, and that 53% have been found only once [bib_ref] Marine natural products: a wave of new drugs?, Montaser [/bib_ref]. Complementary studies investigating the distribution of natural products in chemical space has shown clearly that marine natural products have the broadest distribution, covering many drugrelevant areas [bib_ref] Clustered distribution of natural product leads of drugs in the chemical space..., Tao [/bib_ref]. As such, the focus has recently shifted to marine natural product bioprospecting, which has delivered remarkably high hit rates [bib_ref] Lessons from the past and charting the future of marine natural products..., Gerwick [/bib_ref] [bib_ref] Marine natural products, Blunt [/bib_ref]. The ocean harbors a number of ecological niches and has proven to be home to more microorganisms than any other environment. Considering that 70% of our planet's surface is covered by the oceans, it is not surprising that certain marine ecosystems harbor much higher biological and chemical diversity than what is found terrestrially. Furthermore, the sedentary lifestyle of many of the organisms necessitates a chemical means of defense, and as such natural products are produced as chemical weapons which have evolved into highly effective inhibitors. Since the released compounds become rapidly diluted, marine natural products tend to be highly potent in order to be effective [bib_ref] Drugs form the deep: marine natural products as drug candidates, Haefner [/bib_ref]. The rich biodiversity contained within the oceans (15 animal phyla exclusive to the oceans) makes them a unique and rich drug discovery reservoir [bib_ref] Trends in the discovery of new marine natural products from invertebrates over..., Leal [/bib_ref]. Marine natural product discovery was initially focused on the easily accessible macro-organisms (such as algae, soft corals, and sponges) from which a range of bioactive compounds have been described [bib_ref] Contributions to the study of marine products. XXXII. The nucleosides of sponges, Bergmann [/bib_ref] [bib_ref] Ziconotide: a review of its pharmacology and use in the treatment of..., Mcgivern [/bib_ref] [bib_ref] Statistical research on marine natural products based on data obtained between, Hu [/bib_ref] [bib_ref] Trends in the discovery of new marine natural products from invertebrates over..., Leal [/bib_ref]. However, efforts have gradually turned to the smaller forms of life such as bacteria and fungi [bib_ref] Lessons from the past and charting the future of marine natural products..., Gerwick [/bib_ref] which constitute a large portion of the marine biomass [bib_ref] Microbial diversity in the deep sea and the underexplored "rare biosphere, Sogin [/bib_ref]. Considering the enormous number of microbes, their vast metabolic diversity and the rate of mutations during the past 3.5 billion years, it is expected that there are high levels of genetic and phenotypic variation in marine environments [bib_ref] Microbial diversity in the deep sea and the underexplored "rare biosphere, Sogin [/bib_ref]. Furthermore, marine microorganisms live in a biologically competitive environment with unique, harsh, and fluctuating conditions. They encounter enormous physical and chemical variability including low temperature, high pressure, oligotrophy, high salinity and other competitive environments, and are especially rich in chlorine and bromine elements. Global scale analyses of bacterial diversity identify environment salinity and temperature as the major determinants of microbial community composition, resulting in distinct marine microbiota being selected [bib_ref] Global patterns in bacterial diversity, Lozupone [/bib_ref]. Biofilm formation is a crucial aspect where cell densities are typically 100-1000 fold higher in a biofilm assemblage than in the surrounding water column [bib_ref] The second skin: ecological role of epibiotic biofilms on marine organisms, Wahl [/bib_ref]. Furthermore, the increased competition amongst organisms is thought to be the source of higher production levels of secondary metabolites [bib_ref] Secondary metabolites produced by the marine bacterium Halobacillus salinus that inhibit quorum..., Teasdale [/bib_ref]. In contrast to typical terrestrial environments, marine environments have a very high bacterial diversity at the higher taxonomic levels and a global biogeographical study has shown that there is no more than 12% taxon overlap between bacterial assemblages within and between habitat types [bib_ref] Global patterns in the biogeography of bacterial taxa, Nemergut [/bib_ref]. As a result marine microorganisms represent a unique source of genetic information and biosynthetic capacity which translates to huge chemical diversity. ## Marine microbial natural products Marine microorganisms produce a vast variety of secondary metabolites which could be used to supply the starved pharmaceutical market. Microbial natural products have notable potent therapeutic activities, and also often possess the desirable pharmacokinetic properties required for clinical development [bib_ref] Improving drug discovery from microorganisms, Farnet [/bib_ref]. More than half of the known natural products with anti-microbial, anti-tumor [bib_ref] Lithistid sponges: star performers or hosts to the stars, Bewley [/bib_ref] [bib_ref] Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source,..., Feling [/bib_ref] [bib_ref] Structure and activity of largazole, a potent antiproliferative agent from the Floridian..., Taori [/bib_ref] [bib_ref] Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic..., Rath [/bib_ref] or anti-viral activity are of bacterial origin [bib_ref] Bioactive microbial metabolites-a personal view, Berdy [/bib_ref]. Additional categories include anti-parasitic [bib_ref] Evaluation and development of spinosyns to control ectoparasites on cattle and sheep, Kirst [/bib_ref] [bib_ref] Dermacozines, a new phenazine family from deep-sea dermacocci isolated from a Mariane..., Abdel-Mageed [/bib_ref] , anti-nematodal [bib_ref] Marine natural products and their potential applications as anti-infective agents, Donia [/bib_ref] , anti-inflammation, and neurological [bib_ref] Identification of the putative bryostatin polyketide synthase gene cluster from "Candidatus Endobugulasertula",..., Sudek [/bib_ref]. Pharmaceutically relevant natural products belong to different chemical classes that differ not only in structure, but also in the mechanisms by which they are synthesized. The molecular classes which become pharmaceuticals tend to be alkaloids, terpenoids, polyketides and small peptides, and a wide range of bioactive properties are observed within each class [bib_ref] Antimicrobial activity of heterotrophic bacterial communities from the marine sponge Erylus discophorus..., Graça [/bib_ref]. Furthermore, the elucidation of novel hybrid compounds is providing deeper insights into fascinating enzyme assemblies and mechanisms behind the diversity in structure and biological functions observed in these compounds. Some marine derived microbial examples can be found in the following references: alkaloids [bib_ref] Antioxidant and anti-protease activities of diazepinomicin from the sponge-associated Micromonospora strain RV115, Abdelmohsen [/bib_ref] ; terpenoids [bib_ref] Diversity of the biosynthesis of the isoprene units, Kuzuyama [/bib_ref] [bib_ref] Azamerone, a terpenoid phthalazinone from a marine-derived bacterium related to the genus..., Cho [/bib_ref] [bib_ref] Bioactive compounds from marine actinomycetes, Solanki [/bib_ref] ; polyketides [bib_ref] Antitumor compounds from marine Actinomycetes, Olano [/bib_ref] [bib_ref] Hyaluromycin, a new hyaluronidase inhibitor of polyketide origin from marine Streptomyces sp, Harunari [/bib_ref] , peptides [bib_ref] Antineoplastic agents. Isolation and structure elucidation of bacillistatins 1 and 2 from..., Pettit [/bib_ref] [bib_ref] Sonorensin: an antimicrobial peptide, belonging to the heterocycloanthracin subfamily of bacteriocins, from..., Chopra [/bib_ref] ; and hybrids [bib_ref] Neomarinone, and new cytotoxic marinone derivatives, produced by a marine filamentous bacterium..., Hardt [/bib_ref] [bib_ref] Salinosporamide A: a highly cytotoxic proteasome inhibitor from a novel microbial source,..., Feling [/bib_ref] [bib_ref] Marine natural products, Blunt [/bib_ref]. An additional attraction of microbially derived natural products is that they offer an answer to the supply problem, a major bottleneck in the drug discovery pipeline. The progression of many marine natural products with promising pharmaceutical relevance into clinical phases are halted since the clinical trial stage requires a considerable amount of drug mass; usually kilogram amounts [bib_ref] Bacterial production of the tunicate-derived antitumor cyclic depsipeptide didemnin B, Tsukimoto [/bib_ref]. Most pharmaceutically interesting compounds are found in minute amounts, therefore bioprospecting cannot rely on wild-harvesting as it could lead to the extinction of marine species. More economically feasible, environmentally friendly, and sustainable sources of lead compounds are required. Microbial-based production of lead compounds therefore offers a sustainable solution through the use of culturable marine microorganisms (microbial fermentation). Marine bacteria can respond positively during scaling up processes, and can incorporate sustainable chemical processes for faster establishment of a pilot plant for production (Abd [bib_ref] Response surface methodology for optimising the culture conditions for eicosapentaenoic acid production..., Elrazak [/bib_ref]. The current industrial process for the production of Yondelis, for example, involves the fermentation of Pseudomonas fluorescens for the production of the starting material cyanosafracin B, followed by semi-synthesis to generate the final drug [bib_ref] Synthesis of ecteinascidin ET-743 and phthalascidin Pt-650 from cyanosafracin B, Cuevas [/bib_ref]. Furthermore, strain intensification and elicitation to improve expression are possible through metabolic engineering, as well as the unlocking of untapped cryptic biosynthetic pathways through heterologous host expression [bib_ref] Escherichia coli as a cell factory for heterologous production of nonribosomal peptides..., Li [/bib_ref]. ## Marine metagenomics There is remarkable potential harbored within microorganisms to produce diverse drug-like small molecules for a wide range of applications. The impact and possible success of a single new discovery distinguishes natural products from all other sources of chemical diversity [bib_ref] Improving drug discovery from microorganisms, Farnet [/bib_ref]. However, traditional culture-based approaches used to identify microbial metabolites likely miss the vast majority of bacterial natural products. Only about 1% of bacteria are cultured in vitro and of the approximately 61 bacterial phyla known, 31 lack cultivable representatives [bib_ref] Strategies for culture of 'unculturable'bacteria, Vartoukian [/bib_ref]. Seawater bacteria have a 10-fold lower representation of cultured isolates compared to other environments [bib_ref] Phylogenetic identification and in situ detection of individual microbial cells without culturing, Amann [/bib_ref]. Therefore, if the natural products discovered from cultured marine bacteria are an indication of the diversity available, culture-independent approaches are expected to more successfully access the untapped reservoir of chemical diversity and contribute many more novel marine-derived discoveries. The study of DNA obtained directly from an environmental sample (metagenomics) accesses the collective genomes and bioactive potential of bacterial consortia [bib_ref] Metagenomics: application of genomics to uncultured microorganisms. Microbiol, Handelsman [/bib_ref]. Metagenomics therefore provides a means of exploring novel metabolites from bacteria that are known to be present in marine environments but which remain recalcitrant to culturing [bib_ref] Recent application of metagenomic approaches toward the discovery of antimicrobials and other..., Banik [/bib_ref]. Moreover, metagenomics is particularly attractive for natural product discovery because the genetic information encoding the activities of interest are generally clustered on bacterial genomes, making it possible to clone an entire pathway on an individual or at least a small number of overlapping library clones [bib_ref] Molecular biological access to the chemistry of unknown soil microbes: a new..., Handelsman [/bib_ref] [bib_ref] Recent application of metagenomic approaches toward the discovery of antimicrobials and other..., Banik [/bib_ref]. Therefore, high throughput metagenomic screening approaches, using both sequence-based and functionbased screening, can be employed, in theory, to de-replicate known pathways and compounds and reduce the high degree of redundancy obtained through traditional culture based approaches. Metagenomic screening approaches cover a large range of techniques and are subject to the specifications of the target compound. The particular focus of this review is to evaluate the impact of function-guided strategies as a tool in marine natural product discovery. Specifically, we compare two different functional approaches and their contributions to unlocking the natural product potential harbored in marine microbial genomes. ## Classic functional metagenomic screening In natural product discovery, classical functional screening involves the generation and subsequent screening of metagenomic libraries for the direct detection of the metabolite's properties (e.g., antibacterial, antifungal, antitumor, antiviral activity; Rocha-Martin et al., 2014; . Using whole cells, the culture supernatant or cell pellet extract, this screening approach has been employed with some success. One of the simplest strategies is to test for growth inhibition against a test microbe in top agar overlay assays. This has led to the characterization of a variety of new antibiotics from soil-derived environments [bib_ref] Recombinant environmental libraries provide access to microbial diversity for drug discovery from..., Curtois [/bib_ref] , but no marine-derived studies have been reported, to our knowledge. A more typical approach to functional screening is to screen for a readily detectable phenotype which is representative of the desired bioactive compound, either through the visual detection of pigment production or the use of chromogenic and fluorogenic enzyme substrates which allow the detection of specific catalytic functions encoded on individual clones when incorporated into the growth medium [bib_ref] Metagenomis fro mining new genetic resources of microbial communities, Ferrer [/bib_ref] [bib_ref] Synthetic biology approaches to improve biocatalyst identification in metagenomic library screening, Guazzaroni [/bib_ref]. The antibacterial pigments violacein [bib_ref] Cloning and heterologous expression of a natural product biosynthetic gene cluster from..., Brady [/bib_ref] , indigo [bib_ref] Characterization of a forest soil metagenome clone that confers indirubin and indigo..., Lim [/bib_ref] , and turbomycins [bib_ref] Isolation of antibiotics turbomycina and B from a metagenomic library of soil..., Gillespie [/bib_ref] have been isolated from soil metagenomic libraries. Success with marine libraries; however, has not been reported. A number of other function based screens have yielded a range of different bioactive compounds or activities. Although these screens have not been employed in marine library screening, they are worthy of mention because we expect it is only a matter of time before they are reported. An acylhomoserine lactone synthase promoter fused to a lacZ reporter has been employed to identify AHL lactonases capable of inhibiting Pseudomonas aeruginosa biofilms [bib_ref] Metagenome-derived clones encoding two novel lactonase family proteins involved in biofilm inhibition..., Schipper [/bib_ref]. A phosphopantetheinyl transferase (PPTase)-targeting functional screen has resulted in the efficient recovery of natural product gene clusters from metagenomic libraries [bib_ref] A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product..., Owen [/bib_ref]. Non-ribosomal peptide synthetase and polyketide synthase (PKS) enzymes are activated by PPTases, therefore these enzymes are frequently associated with secondary metabolite gene clusters [bib_ref] Secondary metabolic gene clusters: evolutionary toolkits for chemical innovation, Osbourn [/bib_ref] [bib_ref] A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product..., Owen [/bib_ref]. There is a much greater chance of detecting the expression of a single intact gene than an entire biosynthetic operon, therefore focusing on only a single gene target for the recovery of NRPS and PKS gene clusters FIGURE 1 | A comparison of two function-driven approaches to employ metagenomics for the discovery and production of pharmaceutically relevant marine natural products. Classic functional metagenomic screening: metagenomic libraries are generated in a suitable host and activity screened in a variety of ways, to detect clones expressing metabolites with potential therapeutic properties. The active clones are sequenced to determine the biosynthetic pathway. For certain classes of secondary metabolites, sequence from overlapping clones may be required to compose the entire pathway. The structure of the expressed metabolite is elucidated, following chemical dereplication and characterization methods. If the metabolite is novel, further functional characterization is conducted to evaluate its therapeutic potential. Targeted metagenomic screening: these strategies are guided by traditional chemistry and structure/function-based discoveries in which novel natural products are first isolated and characterized directly from the marine organism or environment. Guided by the chemical classification, a targeted sequence-based analysis can be employed to identify whether the metabolite is microbially encoded, and to subsequently describe the biosynthetic gene cluster. This approach has been employed successfully (detailed in text) when integrated with a number of technologies such as in situ hybridization, single-cell sorting, and whole genome amplification (WGA). The sequence-based analysis of the metagenomic DNA can include gene-targeting using degenerate PCR amplification; or next generation sequencing of the clone library or of the metagenomic DNA directly (shotgun). Where sufficient sequence information is assembled, full genome information can be used to describe novel and uncultured bacteria. The elucidation of the genetic clusters provides the foundation for direct production of the pharmaceutical drug and new analogs through metabolic engineering, and opens the possibility to produce the drugs through heterologous expression. Frontiers in Microbiology | www.frontiersin.org by association increases the chances of identifying "hits" [bib_ref] A functional screen for recovery of 4'-phosphopantetheinyl transferase and associated natural product..., Owen [/bib_ref]. Function-driven screening strategies offer significant advantages to sequence/homology based screening [bib_ref] Metagenomic gene discovery: how far have we moved into novel sequence space?, Tuffin [/bib_ref] [bib_ref] Marine metagenomics: new tools for the study and exploitation of marine microbial..., Kennedy [/bib_ref] [bib_ref] Targeted metagenomics: a high-resolution metagenomics approach for specific gene clusters in complex..., Suenaga [/bib_ref]. This is primarily due to the fact that prior knowledge of the gene sequence for the target activity of interest is not needed, and as a result it is expected that functional screening increases the 'novelty' hit rate. This increases the potential of identifying entirely new classes of genes for both known and novel functions [bib_ref] Metagenomics as advanced screening methods for novel microbial metabolite, Sharma [/bib_ref]. Furthermore, the hits obtained represent an "insurance policy"; guaranteed success of expression in the heterologous host, enabling one to screen for particular properties and under specified conditions, as well as facilitating downstream analyses. The dearth of marine natural product discoveries through functional metagenomics is puzzling considering the increased research focus on marine microorganisms over the last decade [bib_ref] Marine metagenomics: new tools for the study and exploitation of marine microbial..., Kennedy [/bib_ref]. We propose two major reasons for this, (i) heterologous expression challenges and (ii) the sequence technology boom. ## Challenges associated with classic functional metagenomics Natural product discovery, using metagenomics, faces a number of significant challenges and limitations when employing classic functional screening approaches [bib_ref] Marine metagenomics: new tools for the study and exploitation of marine microbial..., Kennedy [/bib_ref] [bib_ref] Escherichia coli as a cell factory for heterologous production of nonribosomal peptides..., Li [/bib_ref] [bib_ref] Emerging concepts promising new horizons for marine biodiscovery and synthetic biology, Reen [/bib_ref]. The most well-known are those associated with heterologous gene expression. [bib_ref] Quantifying the accessibility of the metagenome by random expression cloning techniques, Gabor [/bib_ref] estimated using in silico analysis that only 40% of enzymatic activities can be identified by random cloning of environmental DNA in Escherichia coli. Many studies have highlighted heterologous expression as an enormous challenge limiting the robustness of metagenomics to fully access metabolic potential [bib_ref] Metagenomis fro mining new genetic resources of microbial communities, Ferrer [/bib_ref] [bib_ref] Functional metagenomics for enzyme discovery: challenges to efficient screening, Uchiyama [/bib_ref] [bib_ref] Emerging concepts promising new horizons for marine biodiscovery and synthetic biology, Reen [/bib_ref]. In natural product discovery, these challenges are augmented for a number of reasons. (i) Unlike for other biotechnologically important enzymes and activities typically screened in metagenomic studies, such as the glycosyl hydrolases for example, the activities encoded by particularly the PKS and NRPS pathways, require optimal induction conditions of many genes for expression. The enzymatic megacomplexes for dedicated synthesis of their cognate products are encoded by massive gene clusters, some composed of over 20 genes which are distributed between multiple polycistronic transcriptional units [bib_ref] Engineered polyketide biosynthesis and biocatalysis in Escherichia coli, Gao [/bib_ref] [bib_ref] Secondary metabolic gene clusters: evolutionary toolkits for chemical innovation, Osbourn [/bib_ref]. Obviously there is a much lower chance of expressing an entire biosynthetic pathway in any given heterologous host than a single active enzyme. Secondary metabolite pathways are regulated by pathway specific proteins as well as global regulatory elements in response to changes in nutrient conditions or environmental signals [bib_ref] The regulation of the secondary metabolism of Streptomyces: new links and experimental..., Van Wezel [/bib_ref]. The extremely diverse marine specific factors responsible for unique biochemistries are difficult to replicate in functional screening. For example, it is well-understood that many secondary metabolite pathways expressed in their natural environmental conditions remain silent under laboratory conditions [bib_ref] Marine natural products: a wave of new drugs?, Montaser [/bib_ref] , and this is magnified in heterologous systems. The synergies associated with complex symbiotic and competitive interactions cannot easily be incorporated in simple expression systems. (ii) Even if heterologous expression of a particular pathway is successful, it may not necessarily produce the same compound. Only one isomer may be active and not the other due to the requirement of intermediate compound(s) from the original host or environment [bib_ref] Sponge-associated microorganisms: evolution, ecology, and biotechnological potential. Microbiol, Taylor [/bib_ref] [bib_ref] Antiviral lead compounds from marine sponges, Sagar [/bib_ref]. Furthermore, the absence of a required post-translational modification process, the requirement of in trans genetic elements or the fragmentation of previously clustered genes would not allow functional detection [bib_ref] Defensive bacteriome symbiont with a drastically reduced genome, Nakabachi [/bib_ref]. The use and development of alternative bacterial hosts, expression systems, and multi-host shuttle vectors is crucial to overcoming the limitations discussed. The ability to screen using alternative transcriptional machinery and promoter recognition capabilities should broaden the spectrum of gene expression. Recently, in order to achieve good heterologous expression of novel bioactive compounds, the development of marine-derived hosts such as actinomycete, cyanobacteria, and symbiotic fungi was undertaken to optimize heterologous production [bib_ref] Emerging strategies and integrated systems microbiology technologies for biodiscovery of marine bioactive..., Rocha-Martin [/bib_ref]. The ability to replicate in multiple hosts enables the screening to be conducted in the background of different regulatory and metabolic networks. Furthermore, biosynthetic pathways have also been shown to result in different phenotypes when expressed in different hosts [bib_ref] Expanding smallmolecule functional metagenomics through parallel screening of broad-hostrange cosmid environmental DNA..., Craig [/bib_ref]. (iii) Owing to the large sizes of the biosynthetic pathways, which routinely approach 100 kb, functional screening of metagenomic libraries for the encoded activity is restricted by the need for the entire cluster to be recovered on a single clone . Libraries therefore need to be prepared in bacterial artificial chromosomes (BACs), which can be maintained at low copy number and can carry DNA inserts as large as 350 kb [bib_ref] Cloning and stable maintenance of 300 kilobase-pair fragments of human DNA in..., Shizuya [/bib_ref]. However, it is a major technical challenge to preserve the large size of the metagenomic DNA while sufficiently removing impurities that inhibit cloning. In practice, metagenomic BAC libraries only manage 40 kb insert sizes and rarely greater than 70-100 kb [bib_ref] Molecular biological access to the chemistry of unknown soil microbes: a new..., Handelsman [/bib_ref] [bib_ref] size does matter: application-driven approaches for soil metagenomics, Kakirde [/bib_ref]. Furthermore, metagenomes representing symbiotic communities associated with marine invertebrates represent hundreds of individual genomes. To adequately represent each one requires massive DNA libraries, in the order of 10 6 clones, to be constructed and screened [bib_ref] Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides, Freeman [/bib_ref]. Therefore, metagenome libraries generally vastly underrepresent the true diversity, which has so far prohibited the realization of a functional metagenomic approach [bib_ref] Polyketide assembly lines of uncultivated sponge symbionts from structure-based gene targeting, Fisch [/bib_ref]. (iv) Activities which are initially identified and associated with a library clone extract are sometimes lost before the chemical structure can be determined due to strong negative selection in the heterologous system [bib_ref] Recombinant environmental libraries provide access to microbial diversity for drug discovery from..., Curtois [/bib_ref]. (v) Microbial-derived compounds often have multiple activities; for example anti-tumor [bib_ref] Synthesis, antitumor and antibacterial activities of some novel tetrahydrobenzo, Abbas [/bib_ref] [bib_ref] Novel 1,3,4-oxadiazole thioester derivatives targeting thymidylate synthase as dual anticancer/antimicrobial agents, Du [/bib_ref] , anti-inflammatory [bib_ref] Dual evaluation of some novel 2-amino-substituted coumarinylthiazoles as anti-inflammatory-antimicrobial agents and their..., Chandak [/bib_ref] , and antiprotozoan [bib_ref] Dermacozines, a new phenazine family from deep-sea dermacocci isolated from a Mariane..., Abdel-Mageed [/bib_ref] compounds also display antibacterial activity which may be toxic to the heterologous host. A large proportion of sought-after activities will therefore never be represented in metagenomic libraries. This cannot necessarily be overcome by the use of shuttle vectors because it is in the initial library construction phase that the clones harboring toxic activities will be lost. Ideally metagenomic libraries constructed in shuttle vectors need to be transformed/transfected into the multiple hosts; however, the levels of efficiency required are difficult to generate in non-E. coli hosts. Maintaining low copy numbers may enable the host to survive the toxicity; however, it is highly likely that the screening method will not be sensitive enough to detect the active clone. ## The sequence boom To overcome some of the challenges associated with functional screening, sequence/homology-based screening has been employed in a number of different ways. It is not the intention of this review to compare function vs. sequence based metagenomic methods; however, a brief review is presented to put into context the need for continued attention to functional metagenomic tools. Metagenomic DNA or clone libraries can be screened using degenerate PCR primers designed to conserved sequences within biosynthetic gene clusters [bib_ref] Recent application of metagenomic approaches toward the discovery of antimicrobials and other..., Banik [/bib_ref]. The clustering of biosynthetic genes on a contiguous region of DNA makes homology-based screening attractive. The domain architecture of PKSs and NRPSs in most cases mirrors the structure of the assembled metabolite [bib_ref] Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge..., Piel [/bib_ref]. Therefore, the use of degenerate primers is routinely and successfully employed to first detect conserved NRPS and PKS motifs, followed by the recovery of the remainder of the biosynthetic enzymes by association [bib_ref] On the presence of peptide synthetase and polyketide synthase genes in the..., Moffitt [/bib_ref] [bib_ref] Biomedicinals from the phytosymbionts of marine invertebrates: a molecular approach, Dunlap [/bib_ref] [bib_ref] Genomic mining for novel FADH2-dependent halogenases in marine sponge-associated microbial consortia, Bayer [/bib_ref]. Furthermore, the identification of relatives of known biosynthetic variants could be a strategy to identify or synthesize new structural variants to provide compounds with improved pharmacological properties [bib_ref] Recent application of metagenomic approaches toward the discovery of antimicrobials and other..., Banik [/bib_ref]. However, in some cases up to 99% of the genes detected through PCR screening can represent dominant sequences which are already known and alternative strategies are required to overcome the presence of similar sequences [bib_ref] Antitumor polyketide biosynthesis by an uncultivated bacterial symbiont of the marine sponge..., Piel [/bib_ref] [bib_ref] Metagenomic analysis reveals diverse polyketide synthase gene clusters in microorganisms associated with..., Schirmer [/bib_ref] [bib_ref] Widespread occurrence and genomic context of unusually small polyketide synthase genes in..., Fieseler [/bib_ref] [bib_ref] Diversity of microbes associated with the marine sponge, Haliclona simulans, isolated from..., Kennedy [/bib_ref] [bib_ref] Linking chemical and microbial diversity in marine sponges: possible role for poribac-teria..., Hochmuth [/bib_ref] [bib_ref] PKS and NRPS gene clusters from microbial symbiont cells of marine sponges..., Siegl [/bib_ref] [bib_ref] Diversity of nonribosomal peptide synthetase genes in the microbial metagenomes of marine..., Pimentel-Elardo [/bib_ref] [bib_ref] Polyketide genes in the marine sponge Plakortis simplex: a new group of..., Sala [/bib_ref]. Exciting advancements in next generation DNA sequencing and bioinformatics technologies now negates the need to prepare and sequence clone-libraries. Shotgun metagenomic sequencing has made it possible to rapidly identify large biosynthetic gene clusters and subsequent predictions of their chemical structure can be made [bib_ref] NORINE: a database of nonribosomal peptides, Caboche [/bib_ref] [bib_ref] Diversity of monomers in nonribosomal peptides: towards the prediction of origin and..., Caboche [/bib_ref] [bib_ref] NRPSpredictor2. A web server for predicting NRPS adenylation domain specificity, Röttig [/bib_ref] [bib_ref] Computational tools for the synthetic design of biochemical pathways, Medema [/bib_ref] [bib_ref] Pep2Path: automated mass spectrometry-guided genome mining of peptidic natural products, Medema [/bib_ref] [bib_ref] antiSMASH 2.0. A versatile platform for genome mining of secondary metabolite producers, Blin [/bib_ref]. While purely in silico approaches are generally limited to the detection of one or more well-characterized gene cluster classes [bib_ref] Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene..., Cimermancic [/bib_ref] , continued developments in bioinformatics pipelines and other technologies are already improving access to diverse and novel secondary metabolite genes and clusters, including providing access to the "rare biosphere" (we refer readers to a number of examples: [bib_ref] Catalytic promiscuity in the biosynthesis of cyclic peptide secondary metabolites in planktonic..., Li [/bib_ref] [bib_ref] Antiviral lead compounds from marine sponges, Sagar [/bib_ref] [bib_ref] Polyketide synthase gene diversity within the endemic sponge Arenosclera brasiliensis microbiome, Trindade-Silva [/bib_ref] [bib_ref] Deep sequencing of non-ribosomal peptide synthetases and polyketide synthases from the microbiomes..., Woodhouse [/bib_ref] [bib_ref] Insights into secondary metabolism from a global analysis of prokaryotic biosynthetic gene..., Cimermancic [/bib_ref]. Furthermore, the deposition of more functionally curated sequence data in publically available databases should improve the ability to use purely bioinformatics based screening for the identification of novel gene clusters [bib_ref] Metagenomic gene discovery: how far have we moved into novel sequence space?, Tuffin [/bib_ref] [bib_ref] Targeted metagenomics: a high-resolution metagenomics approach for specific gene clusters in complex..., Suenaga [/bib_ref]. In silico approaches facilitate rapid dereplication of common biosynthesis clusters and thus the prioritization of new chemical scaffolds for experimental characterization. Although, targeted induction in heterologous expression systems has delivered some success from the marine environment [bib_ref] Shotgun cloning and heterologous expression of the patellamide gene cluster as a..., Long [/bib_ref] [bib_ref] Linking chemical and microbial diversity in marine sponges: possible role for poribac-teria..., Hochmuth [/bib_ref] [bib_ref] Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic..., Rath [/bib_ref] [bib_ref] Direct capture and heterologous expression of Salinispora natural product genes for the..., Bonet [/bib_ref] [bib_ref] Directed natural product biosynthesis gene cluster capture and expression in the model..., Li [/bib_ref] , it is not easily going to deliver compounds with the sought after properties required by the pharmaceutical markets in a high throughput manner, when taking a purely in silico discovery route. For example, the swf cluster, a new mono-modular type I PKS/FAS (fatty acid synthase) was identified through screening of the Plakortis simplex sponge metagenome (Della [bib_ref] Polyketide genes in the marine sponge Plakortis simplex: a new group of..., Sala [/bib_ref]. The entire pathway was expressed in E. coli; however, the production of an associated metabolite was not detected. Notwithstanding all the difficulties associated with heterologous expression and the inability to conduct this in a high throughput manner, novel sequence will not necessarily result in the pharmaceutically required biological properties. It is currently easier and cheaper to generate vast volumes of gene and genome sequence information than it is to produce the experimental characterizations, and the gap between these is growing rapidly [bib_ref] Functional assignment of metagenomic data: challenges and applications, Prakash [/bib_ref] [bib_ref] Next generation sequencing and bioinformatic bottlenecks: the current state of metagenomic data..., Scholz [/bib_ref] [bib_ref] Current opportunities and challenges in microbial metagenome analysis-A bioinformatic perspective, Teeling [/bib_ref] [bib_ref] Emerging concepts promising new horizons for marine biodiscovery and synthetic biology, Reen [/bib_ref]. ## Targeted metagenomic strategies in marine discovery From a pharmaceutical point of view, marine drug discovery necessitates a focus on functionality. Irrespective of the approach employed, obtaining biologically active and pure compounds with the desired activity or properties is the end goal. The ability to achieve this through function-driven screening strategies is, in principle, the golden standard. Given the limitations discussed above this will remain a major challenge. Relative to other environmental biodiscovery efforts, classical functional metagenomic screening of marine sources has yet to contribute significantly to the pharmaceutical industry. However, significant improvements in the chemical and genetic sciences and the integration of these technologies, has resulted in a number of successes which are beginning to drive the development of parallel technologies. Instead of functionally screening a metagenome clone library, a targeted approach which harnesses prior knowledge of marine natural product diversity, chemistry, and biological activity is bridging the gap between the accumulation of microbial genetic datasets and functional and ecological relevance . In this section we highlight some of the recent discoveries that have employed metagenomic strategies which were guided primarily by initial structural and functional characteristics and associated pharmaceutical interest. ## Bryostatins Bryostatin 1, a polyketide initially detected in 1968 in extracts from the marine bryozoan Bugula neritina [bib_ref] The bryostatins, Pettit [/bib_ref] , raised interest due to its cytotoxic activity against multiple carcinomas, with proteinase kinase C as its molecular target [bib_ref] The odyssey of marine pharmaceuticals: a current pipeline perspective, Mayer [/bib_ref]. Bryostatin 1 has been tested in over 80 clinical trials for cancer and is also being assessed in Phase I trials as an anti-Alzheimer's drug. Although the in vivo activity was initially detected directly from the bryozoan, it was for many years suspected that the compound was produced by a bacterial symbiont since a difference in the types of bryostatins found in B. neritina correlated with genetically different bacterial symbionts [bib_ref] Identification of sibling species of the bryozoan Bugula neritina that produce different..., Davidson [/bib_ref]. A particular symbiont in the larvae of the bryozoan was identified and suspected to be the producer, and was proposed as a novel gammaproteobacterium, 'Candidatus Endobugula sertula.' Attempts to separate the bacterial cells from the host as a way to confirm Ca E. sertula as the producer of the bryostatin were inconclusive, therefore a metagenomic approach was employed [bib_ref] Evidence for the biosynthesis of bryostatins by the bacterial symbiont "Candidatus Endobugula..., Davidson [/bib_ref]. Since, bryostatin is a type I polyketide, PKSbased screening was conducted and led to the confirmation that the genes coding for type I PKS complex were derived from the symbiotic population. Further query involving the growth of B. neritina colonies after antibiotic treatments and in situ hybridization experiments confirmed that "E. sertula" was the source of the bryostatins. A cosmid library was prepared from B. neritina Mission Bay metagenomic DNA, and was screened by hybridization [bib_ref] bryA: an unusual modular polyketide synthase gene from the uncultivated bacterial symbiont..., Hildebrand [/bib_ref] using a β-ketoacyl synthase probe identified previously [bib_ref] Evidence for the biosynthesis of bryostatins by the bacterial symbiont "Candidatus Endobugula..., Davidson [/bib_ref]. Several overlapping clones were sequenced revealing the 65 kb bry gene cluster [bib_ref] bryA: an unusual modular polyketide synthase gene from the uncultivated bacterial symbiont..., Hildebrand [/bib_ref]. Probes spanning the bry gene cluster were hybridized to 'Candidatus E. sertula'-enriched DNA to confirm the symbiont as the origin of the gene cluster. Further interrogation in two closely related "E. sertula" strains from different host species identified two different gene cluster arrangements [bib_ref] Identification of the putative bryostatin polyketide synthase gene cluster from "Candidatus Endobugulasertula",..., Sudek [/bib_ref]. In one strain the gene cluster is contiguous, while in the other strain the PKS genes are split from the accessory genes. Due to the difficulties in obtaining sufficient supply of the bryostatins, their clinical application occurred decades after their discovery. Since "E. sertula" remains unculturable, heterologous expression of the bry gene cluster could be considered for the production of bryostatins in large enough quantities for pharmaceutical development. ## Et-743 (yondelis r ) Anti-cancer activity in extract from the sea squirt Ecteinascidia turbinata was identified in 1969; however, it was only in 1984 that the structure of one of the compounds, Ecteinascidin 743 (ET-743), was determined [bib_ref] Antitumor compounds from tunicates, Rinehart [/bib_ref]. ET-743 (Yondelis R ) is now an approved anti-cancer agent [bib_ref] Lithistid sponges: star performers or hosts to the stars, Bewley [/bib_ref]. Attempts to farm the sea squirt to provide sufficient supply of the compound had limited success, and it is currently generated in suitable quantities for clinical use by a lengthy semi-synthetic process [bib_ref] Synthesis of ecteinascidin ET-743 and phthalascidin Pt-650 from cyanosafracin B, Cuevas [/bib_ref] [bib_ref] Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic..., Rath [/bib_ref]. The similarity of ET-743 to three other bacterial derived natural products (saframycin A, Streptomyces lavendulae; saframycin Mx1, Myxococcus xanthus; safracin B, Pseudomonas fluorescens; [bib_ref] Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic..., Rath [/bib_ref] suggested that ET-743 was produced by a marine bacterial symbiont. Using metagenomic sequencing of total DNA from the microbial consortium associated with the tunicate resulted in the assembly of a 35 kb contig containing 25 genes encoding a NRPS biosynthetic pathway. Rigorous sequence analysis of two large unlinked contigs suggested that 'Candidatus Endoecteinascidia frumentensis' was the producer of the metabolite. Subsequent metaproteomic analysis confirmed expression of three key biosynthetic proteins. The complete genome of 'Candidatus Endoecteinascidia frumentensis' was very recently determined, showing an extremely reduced genome (∼631 kb) and evidence of an endosymbiotic lifestyle [bib_ref] Identification and analysis of the bacterial endosymbiont specialized for production of the..., Schofield [/bib_ref]. Having the pathway elucidated provides the foundation for direct production of the drug and new analogs through metabolic engineering [bib_ref] Meta-omic characterization of the marine invertebrate microbial consortium that produces the chemotherapeutic..., Rath [/bib_ref]. ## Patellazoles The Lissoclinum patella tunicate has garnered interest due to it representing a rich source of potential bioactive drug leads [bib_ref] Origin and variation of tunicate secondary metabolites, Schmidt [/bib_ref]. The patellazoles were isolated directly from the tunicate in the late 1980s and characterized as a new family of novel thiazole-containing polyketide metabolites [bib_ref] Patellazole B: a novel cytotoxic thiazolecontaining macrolide from the marine tunicate Lissoclinum..., Corley [/bib_ref] [bib_ref] Patellazole C: a novel cytotoxic macrolide from Lissoclinum patella, Zabriskie [/bib_ref]. In addition to their chemical novelty, they gained interest due to their potent cytotoxic activity against human cell lines as well as antifungal (Candida albicans) activity [bib_ref] Patellazole C: a novel cytotoxic macrolide from Lissoclinum patella, Zabriskie [/bib_ref]. The patellamides, also isolated from this tunicate had already been shown to be produced by the cyanobacterial symbiont, Prochloron didemni. Although, P. didemni is the major symbiont, L. patella harbors a complex microbiome [bib_ref] Complex microbiome underlying secondary and primary metabolism in the tunicate-Prochloron symbiosis, Donia [/bib_ref] , and therefore there stood the possibility that the patellazoles were also produced by a symbiont. Due to the multiple acetate units, patellazoles were hypothesized to be produced by a type I PKS pathway, as well as a NRPS module for the incorporation and cyclization of a cysteine unit to generate the thiazole ring. Based on this information an exhaustive sequence based screening of a metagenome clone library prepared from the tunic-cloaca habitats was undertaken, but did not locate the biosynthetic pathway. PCR amplification revealed PKS genes from the trans-acyltransferase family, consistent with patellazole biosynthesis, in the tiny zooids but not in the bulk tunic. DNA extracted from the zooids fraction was subjected to shotgun sequencing and the assembly thereof resulted in a complete genome which contained a 86 kb trans-AT PKS pathway. The predicted biosynthetic model of the encoded pathway was consistent with patellazoles structure, thus strongly supporting the assignment. The assembled genome was considered to belong to an uncultured symbiont, designated as Candidatus "Endolissoclinum faulkneri, " most closely related to free-living marine α-proteobacteria. ## Pederin-led discovery of the onnamides Pederin and mycalamides A and B, encoded by a mixed modular PKS-non-ribosomal peptide synthetase (NRPS) system, are highly active antitumor compounds [bib_ref] The pederin family of antitumor agents: structures, synthesis and biological activity, Narquizian [/bib_ref]. These compounds block mitosis at levels as low as 1 ng/ml by inhibiting protein and DNA synthesis without affecting RNA synthesis [bib_ref] Paederus dermatitis, Singh [/bib_ref]. They prevent cell division, and have been shown to extend the life of cancerous mice. Consequently they have garnered interest as potential anti-cancer treatments [bib_ref] Paederus, sensulato (Coleoptera: Staphylinidae): natural history and medical importance, Kanamitsu [/bib_ref]. These compounds were initially known exclusively from terrestrial Paederus and Paederidus beetles and after many years of speculation were finally shown to be produced by an uncultured symbiotic Pseudomonas associated with the Paederus fuscipes beetles using metagenomic approaches [bib_ref] Evidence for a symbiosis island involved in horizontal acquisition of pederin biosynthetic..., Piel [/bib_ref]. Interestingly, these insects use pederin as a chemical weapon against predators and when in contact with human skin cause severe dermatitis [bib_ref] Paederusfuscipes dermatitis. A histopathological study, Borroni [/bib_ref] [bib_ref] Evidence for a symbiosis island involved in horizontal acquisition of pederin biosynthetic..., Piel [/bib_ref]. Metabolites with high structural similarity to pederin were identified in the marine sponges of the order Lithistida [bib_ref] Lithistid sponges: star performers or hosts to the stars, Bewley [/bib_ref] , many of which exhibit extremely potent antitumor activity and also selectivity against solid tumor cell lines [bib_ref] Psymberin, a potent spongederived cytotoxin from Psammocinia distantly related to the pederin..., Cichewicz [/bib_ref]. A pederin-informed survey of PKS amplicons from the Japanese sponge Theonella swinhoei metagenome, a species with exceptionally rich chemistry [bib_ref] Bioactive sponge peptides, Fusetani [/bib_ref] [bib_ref] Lithistid sponges: star performers or hosts to the stars, Bewley [/bib_ref] , revealed a wide range of distinct KS sequences [bib_ref] Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured..., Piel [/bib_ref]. Three of these belonged to an evolutionarily distinct enzyme family, the trans-acyl-transferase (trans-AT) PKSs, and corresponded to onnamide biosynthesis pathways. These trans-AT PKSs therefore were expected to encode the pederin-like compounds with antitumor activity produced by the sponges. Further, screening of the metagenomes from other T. swinhoei specimens revealed that these trans-AT PKSs could only be detected in the sponges which had previously shown to contain pederin-type compounds, while no amplification was obtained for sponges devoid of these compounds. It has now been confirmed that the onnamides are produced by an unculturable symbiont, 'Candidatus Entotheonella spp.' [bib_ref] An environmental bacterial taxon with a large and distinct metabolic repertoire, Wilson [/bib_ref]. ## Psymberin Psymberin, a highly cytotoxic and selective antitumor polyketide, has been isolated from a number of different marine sponges [bib_ref] The psymberin story-biological properties and approaches towards total and analogue syntheses, Bielitza [/bib_ref]. It took 11 years and 600 samples for the structure of this compound to be assigned. There is immense interest in this natural product due to its complex architecture, biological properties and scarcity in nature. As with FIGURE 2 | A representation of the ubiquity of "Entotheonella" species in taxonomically diverse marine sponges and the notable secondary metabolites they produce. Metabolite structure and function information obtained directly from the Theonella swinhoei and Discodermia calyz sponges informed a targeted metagenomic approach to identify the biosynthetic pathways encoding these metabolites. This ultimately led to the discovery of "Entotheonella," described as "talented producers" due to their chemically diverse metabolism. The full potential of Entotheonella species has yet to be explored. Photos were provided by T. Mori, P. Poppe, and T. Wakimoto. Frontiers in Microbiology | www.frontiersin.org the onnamides, psymberin is a member of the pederin family, also synthesized by a trans-AT PKS [bib_ref] Targeting modular polyketide synthases with iteratively acting acyltransferases from metagenomes of uncultured..., Piel [/bib_ref] , but is distinguished from the other pederins due to its excellent cytotoxicity values which exceeds those of the other family members. A trans-AT PKS PCR screening approach, as described above for the onnamides, was used to elucidate the complete biosynthetic pathway for psymberin from the Psammocinia aff. bulbosa sponge metagenome [bib_ref] Polyketide assembly lines of uncultivated sponge symbionts from structure-based gene targeting, Fisch [/bib_ref]. The genomic region showed typical bacterial architecture, suggesting a bacterial symbiont origin. However, unlike for the pederin and onnamide examples, there were not enough similarities to other bacterial genes to identify the bacterium. ## Polytheonamides The polytheonamides represent another group of exceptionally potent natural product toxins isolated from the Theonella swinhoei sponges, and are particularly noteworthy for their size and structural complexity [bib_ref] Solution structure of polytheonamide B, a highly cytotoxic nonribosomal polypeptide from marine..., Hamada [/bib_ref]. These 48-residue peptides were expected to be products of a non-ribosomal peptide synthetase, since of the 19 different amino acids that constitute these peptides, 13 are nonproteinogenic. Furthermore, the peptides include multiple D-configured and C-methylated residues. However, the size of the NRP biosynthetic machinery required to produce a 48 residue peptide prompted a search for an unusual ribosomal pathway. With the knowledge of the peptide sequence, degenerate PCR primers were designed to the proposed precursor peptide, and used to conduct a semi-nested PCR from a T. swinhoei metagenome [bib_ref] Metagenome mining reveals polytheonamides as posttranslationally modified ribosomal peptides, Freeman [/bib_ref]. Sequenced amplicons revealed codon sequences that precisely corresponded to an unprocessed polytheonamide precursor, not only confirming a ribosomal origin, but also suggesting that it is produced by a bacterial endosymbiont. Further screening of the metagenome library revealed the entire 12 gene biosynthetic pathway. Microscopic analysis of T. swinhoei (Y chemotype) samples identified a highly enriched population of fluorescent filamentous bacteria showing morphological similarity to the symbiont 'Candidatus Entotheonella palauensis, ' the suspected producer of antifungal peptides isolated from a Palauan T. swinhoei chemotype [bib_ref] Identification of the antifungal peptide-containing symbiont of the marine sponge Theonella swinhoei..., Schmidt [/bib_ref]. Using single cell genomics (fluorescence assisted cell sorting and whole genome amplification) combined with pathway specific PCR, the identification of the polytheonamide producer was attributed to an uncultured "Entotheonella" spp. [bib_ref] An environmental bacterial taxon with a large and distinct metabolic repertoire, Wilson [/bib_ref]. Further, screening using onnamide pathway specific markers indicated that the "Entotheonella" spp. were the source of both the onnamide and polytheonamide compounds. ## Calyculin a Calyculin A was originally described in 1986 as a major cytotoxic compound isolated from Discodermia calyx, a marine sponge of the Theonellidae family [bib_ref] Calyculin A, a novel antitumor metabolite from the marine sponge Discodermia calyx, Kato [/bib_ref] , and is to date associated exclusively with marine sponges [bib_ref] Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont, Wakimoto [/bib_ref]. Calyculin A represents a fairly sophisticated and unique structure whose biosynthesis was reminiscent of a polyketide and non-ribosomal peptide hybrid pathway incorporating some remarkable modification processes. Calyculin-related compounds have been isolated from a number of different sponges which hinted toward a symbiont being responsible for its production [bib_ref] Isolation of calyculins, calyculinamides, and swinholide H from the New Zealand deep-water..., Dumdei [/bib_ref] [bib_ref] A new cytotoxic calyculinamide derivative, geometricin A, from the Australian sponge Luffariella..., Kehraus [/bib_ref]. However, it was only very recently that the biosynthetic gene cluster was identified through a metagenomic approach. Based on the initial hypothesis that calyculin A was a type I polyketide, a metagenome library of D. calyx was sequentially screened by PCR amplification using trans-ATtype KS, adenylation domain (NRPS) and HMGS-like motif primers [bib_ref] Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont, Wakimoto [/bib_ref]. Spanning over 150 kb, a gene cluster containing 29 KS and 5 A domains was identified. The collinearity between the order of the modules and the order of the biosynthetic reactions provided strong evidence that the cluster encoded calyculin A synthesis. Using the entire gene cluster as a probe and employing CARD-FISH, as well as laser microdissection, a filamentous bacterium was identified to harbor the calyculin pathway. The 16S rRNA sequence of this bacterium displayed 97% identity to the 'Candidatus Entotheonella factor' isolated from the T. swinhoei sponges. ## From function to genes to species The power of metagenomics to identify novel and pharmaceutically relevant organisms, resulting from first obtaining functional and structural data, has been elegantly represented in the examples discussed above. To demonstrate this further, the "Entotheonella" and the 'Candidatus Endolissoclinum faulkneri' stories are elaborated . Genome sequencing of several of the single cell sorted events in the [bib_ref] An environmental bacterial taxon with a large and distinct metabolic repertoire, Wilson [/bib_ref] study indicated the presence of two closely related "Entotheonella" variants, with 97.6% identical 16S rRNA sequences, and 97% identity to E. palauensis. These are only 82% identical to representatives from known bacterial phyla and form a well-separated clade, and therefore have been proposed to represent a new candidate phylum "Tectomicrobia." Both genomes exceed 9 Mb, representing some of the largest known prokaryote genomes. Analysis of the metabolic genes identified over 28 biosynthetic clusters, encoding ribosomal, polyketide and non-ribosomal peptide biosynthesis. Using bioinformatics based predictions, several of the clusters could be assigned to known bioactive peptides isolated from the Japanese T. swinhoei, and together with tandem mass spectrometry-based molecular networking a high diversity of previously unknown metabolite families were identified. The combination of these properties is so rare that the new phylum to which these isolates have been assigned is considered the successor to the Actinobacteria, the well-known source of the majority of the world's antibiotics and anticancer agents [bib_ref] A talented genus, Jaspars [/bib_ref]. Screening for the distribution of these talented producers indicated that they are geographically widespread and are symbiotically associated with other sponge types [bib_ref] An environmental bacterial taxon with a large and distinct metabolic repertoire, Wilson [/bib_ref]. The discovery of a calyculin producing "Entotheonella" symbiotically associated with D. calyx further expands the number of biosynthetic enzymes and chemical scaffolds encompassed by this genus [bib_ref] Calyculin biogenesis from a pyrophosphate protoxin produced by a sponge symbiont, Wakimoto [/bib_ref] , but also serves to highlight the differences between the "Entotheonella" populations in different sponges. Attempts to culture the producing symbionts have been unsuccessful. Access to the genome sequences should give important insights to the organism's metabolism, and such clues to their physiology could inform on the development of appropriate culturing strategies. Several uncultured symbionts have been successfully isolated using such genome sequence-guided strategies [bib_ref] Genome-based design of a cell-free culture medium for Tropheryma whipplei, Renesto [/bib_ref] [bib_ref] Directed culturing of microorganisms using metatranscriptomics, Bomar [/bib_ref]. In thestudy the patellazole encoding Ca. E. faulkneri genome assembled to a mere 1.48 Mbp and showed extensive genome reduction characteristics. Unlike other bacteria with streamlined genomes, Ca. E. faulkneri has distinguishing features which strongly suggest that it could not exist independently of its host, L. patella. Phylogenetic analysis of patellazole-containing and patellazole-negative tunicates provides evidence that the symbiont has coevolved with the tunicate host and would therefore be transmitted vertically. The patellazole pathway is the only secondary metabolite pathway encoded in the Ca. E. faulkneri genome, and represents >10% of the coding sequence. The maintenance of such a large pathway in a genome that is so streamlined as to eliminate most functions indicates its importance to the symbiotic relationship. However, the patellazoles are highly toxic to eukaryotic cells and are found in high amounts in L. patella, and it is intriguing that the host has apparently adapted to tolerate such high concentrations. Clearly the patellazoles provide important chemical defense to the host which in turn ensures that the symbiont is maintained. Interestingly, Ca. E. faulkneri is found sporadically in L. patella tunicates. Patellazole-positive and negative L. patella collected within 250 m of each other show that Ca. E. faulkneri is only associated with the patellazole-positive colonies, and only in the zooids fraction. This is despite patellazole-positive and negative colonies having nearly identical tunicate phylogeny, and containing virtually identical microbial communities, with the exception of the Ca. E. faulkneri. The exclusive localization of Ca. E. faulkneri in the zooids and only in certain L. patella colonies is intriguing. Considering the L. patella zooids filter feed and excrete waste into the cloacal cavities, this could perhaps provide some leads of investigation to further understanding the Ca. E. faulkneri localization and the symbiotic relationship. These discoveries raise several fundamental biological questions relating to: symbiont and secondary metabolite evolution, mechanisms of natural product symbiosis, the role of the natural products in imparting a direct competitive advantage to individual members of a bacterial consortium, and how these symbiotic interactions contribute to the ecology of the marine environment. However, what is now clearly appreciated is that the genomes of previously uncultured bacteria harbor an unprecedented richness of novel compound diversity, and await discovery. ## Conclusion and future prospects The remarkable exploration of marine organisms and their structurally diverse natural products spans a highly active period of over 40 years [bib_ref] Lessons from the past and charting the future of marine natural products..., Gerwick [/bib_ref]. With attention turning to marine microorganisms as a source of new natural product chemistry, and the realization that many compounds previously isolated are metabolic products of unculturable microbes, marine metagenomics promises to illuminate new bioactivities and chemistries that were previously unattainable. Despite metagenomics being a relatively young technology, it is globally appreciated that major advances are needed given the challenges that now bottleneck future developments, irrespective of whether functional or sequence guided approaches are to be employed. In order to maximize our ability to harvest marine resources the synergic combination of a number of complementary methodologies and integration of functional and informatics approaches will be required [bib_ref] Emerging concepts promising new horizons for marine biodiscovery and synthetic biology, Reen [/bib_ref]. The examples presented, employing a targeted and function-guided strategy, demonstrate how metagenomic technologies have advanced several research disciplines and our understanding of microbial genetic and biological diversity and ecology. Armed with information of the chemical structure and biological activity of pharmaceutically relevant compounds, an informed metagenomic strategy, in combination with in situ hybridization, single cell-sorting, whole genome amplification, and next generation sequencing, has successfully identified novel biosynthetic gene clusters and novel microbes that produce the metabolites. The path that led from similar compounds being found in organisms as divergent as marine sponges and beetles, to the discovery that microorganisms were the producers, and the role metagenomics played, makes a fascinating story demonstrating a perfect blend of fundamental and applied science, exemplifying the power of employing integrated technologies. For marine metagenomics to significantly contribute to delivering pharmaceutically relevant compounds, improvements in, and integration of, various approaches and strategies is key. One of the most important hindrances encountered thus far in natural product research is re-isolation of known compounds. Thus chemical and biological de-replication is a crucial step in the process, and applies to metagenomic guided discovery as well, irrespective of the metagenomic approach employed. While sequence-based metagenomic approaches offer the power of discrimination, the expression of the pathways and the functional and biochemical characterization of the encoded products is crucial. Genome data is being produced at a dizzying pace; however, without focusing on heterologous expression challenges and the development of functional screens our capacity to uncover and develop the next generation of pharmaceutically relevant molecules will be limited [bib_ref] Emerging concepts promising new horizons for marine biodiscovery and synthetic biology, Reen [/bib_ref]. There are two long standing schools of thought on natural products discovery: 'isolate and then test' vs. 'test and then isolate' [bib_ref] Lessons from the past and charting the future of marine natural products..., Gerwick [/bib_ref]. A parallel can be drawn to employing metagenomic tools to natural product discovery: "sequence and then test" vs. "test and then sequence." This review summarizes some of the most recent marine discoveries through the latter approach, born out of traditional chemistry-guided discovery conducted over several decades. However, to maximize our capacity to mine metagenomes for activities which have yet to be identified, parallel developments in a number of technologies need continuous attention; including biological assay screening; isolation and separation methods and analytical chemistry techniques. Peptidogenomics represents a recent advancement in high throughput mass spectrometry (MS; [bib_ref] A mass spectrometry-guided genome mining approach for natural product peptidogenomics, Kersten [/bib_ref] [bib_ref] Mass spectrometry of natural products: current, emerging and future technologies, Bouslimani [/bib_ref] [bib_ref] Pep2Path: automated mass spectrometry-guided genome mining of peptidic natural products, Medema [/bib_ref]. This automated approach iteratively matches the chemotypes of peptide natural products to their biosynthetic gene clusters through de novo tandem MS (MSn) and genome-mining [bib_ref] Emerging concepts promising new horizons for marine biodiscovery and synthetic biology, Reen [/bib_ref]. This constitutes a paradigm shift from the one molecule-per-study approach to drug discovery , and may be the key to revealing novel marine natural products from metagenomes, for advancement into the drug discovery development pipeline. There is no doubt that as yet uncultured bacteria are a rich source of novel bioactive molecules with potent therapeutic activity, and these are exciting times to be a researcher in the field. August 2015 | Volume 6 | Article 890 Frontiers in Microbiology | www.frontiersin.org AcknowledgmentsWe thank the South African National Research Foundation, The Department of Science and Technology (DST) and the University of the Western Cape for financial support. The authors would like to thank T. Mori, P. Poppe, and T. Wakimoto for the photographic contributions as indicated in the figure legends.

Phosphate‐induced resistance to pathogen infection in Arabidopsis In nature, plants are concurrently exposed to a number of abiotic and biotic stresses. Our understanding of convergence points between responses to combined biotic/abiotic stress pathways remains, however, rudimentary. Here we show that MIR399 overexpression, loss-of-function of PHOSPHATE2 (PHO2), or treatment with high phosphate (Pi) levels is accompanied by an increase in Pi content and accumulation of reactive oxygen species (ROS) in Arabidopsis thaliana. High Pi plants (e.g., miR399 overexpressors, pho2 mutants, and plants grown under high Pi supply) exhibited resistance to infection by necrotrophic and hemibiotrophic fungal pathogens. In the absence of pathogen infection, the expression levels of genes in the salicylic acid (SA)-and jasmonic acid (JA)-dependent signaling pathways were higher in high Pi plants compared to wild-type plants grown under control conditions, which is consistent with increased levels of SA and JA in non-infected high Pi plants. During infection, an opposite regulation in the two branches of the JA pathway (ERF1/PDF1.2 and MYC2/VSP2) occurs in high Pi plants. Thus, while pathogen infection induces PDF1.2 expression in miR399 OE and pho2 plants, VSP2 expression is downregulated by pathogen infection in these plants. This study supports the notion that Pi accumulation promotes resistance to infection by fungal pathogens in Arabidopsis, while providing a basis to better understand interactions between Pi signaling and hormonal signaling pathways for modulation of plant immune responses. # Introduction In nature, plants are simultaneously exposed to a combination of biotic and abiotic stresses that are diverse in time and space, which requires proper integration and interactions between different stress response pathways. Exposure to a single stress might, however, impact the plant response to another stress [bib_ref] Enhancing crop resilience to combined abiotic and biotic stress through the dissection..., Kissoudis [/bib_ref] [bib_ref] Plant immune system: crosstalk between responses to biotic and abiotic stresses the..., Nejat [/bib_ref] [bib_ref] MicroRNA400-guided cleavage of pentatricopeptide repeat protein mRNAs renders Arabidopsis thaliana more susceptible..., Pandey [/bib_ref]. For instance, plant immune responses and disease resistance can be altered in plants exposed to drought or high salinity [bib_ref] The interaction of plant biotic and abiotic stresses: from genes to the..., Atkinson [/bib_ref] [bib_ref] Predisposition in plant disease: exploiting the nexus in abiotic and biotic stress..., Bostock [/bib_ref] [bib_ref] Antagonistic interaction between systemic acquired resistance and the abscisic acid-mediated abiotic stress..., Yasuda [/bib_ref]. Inappropriate supply of mineral nutrients (e.g., nitrogen supply) might also impact disease severity [bib_ref] Diversity and genetics of nitrogen-induced susceptibility to the blast fungus in rice..., Ballini [/bib_ref] [bib_ref] The effect of nitrogen on disease development and gene expression in bacterial..., Snoeijers [/bib_ref]. However, the effect of combined abiotic and biotic stress factors might vary depending on the nature of these interactions, and the plant response to simultaneously or sequentially applied stresses cannot be simply inferred from responses to individual stresses [bib_ref] Transcriptome dynamics of Arabidopsis during sequential biotic and abiotic stresses, Coolen [/bib_ref] [bib_ref] The plant immune system in heterogeneous environments, Nobori [/bib_ref] [bib_ref] MicroRNA400-guided cleavage of pentatricopeptide repeat protein mRNAs renders Arabidopsis thaliana more susceptible..., Pandey [/bib_ref] [bib_ref] Simultaneous application of heat, drought, and virus to Arabidopsis plants reveals significant..., Prasch [/bib_ref]. As stress responses are costly, when facing multiple stresses simultaneously, plants need to prioritize their stress responses for efficient use of finite resources, in accordance with the optimal defense theory (ODT) [bib_ref] Defence on demand: mechanisms behind optimal defence patterns, Meldau [/bib_ref]. According to the ODT, stress responses are prioritized in the most valuable parts [bib_ref] Testing the optimal defense hypothesis in nature: variation for glucosinolate profiles within..., Keith [/bib_ref] , and recent findings indicate that Arabidopsis plants spatially separate contrasting stress responses in leaves of different ages (e.g., young leaves exhibit higher biotic stress responses but lower abiotic stress responses compared with old leaves) . To date, little information is available on the molecular mechanisms by which biotic and abiotic stress responses are differentially prioritized in plants and how they adapt to conflicting stresses for optimal responses. To defend themselves against pathogens, plants have evolved an innate immune system in which many interconnected processes are involved . Pathogen-induced pathways are defined principally according to the molecules recognized by the host plant [bib_ref] A renaissance of elicitors: perception of microbe-associated molecular patterns and danger signals..., Boller [/bib_ref] [bib_ref] Regulation of pattern recognition receptor in plants, Couto [/bib_ref] [bib_ref] Of PAMPs and effectors: the blurred PTI-ETI dichotomy, Thomma [/bib_ref] [bib_ref] The coming of age of EvoMPMI: evolutionary molecular plant-microbe interactions across multiple..., Upson [/bib_ref]. Plants recognize pathogen-associated molecular patterns (PAMPs) by receptors at the plasma membrane, which triggers the induction of multiple cascades leading to the induction of immune responses, referred to as PAMP-triggered immunity (PTI). Components of PTI include the reinforcement of cell walls, accumulation of reactive oxygen species (ROS), activation of phosphorylation cascades, production of antimicrobial compounds, and accumulation of pathogenesis-related proteins, among others [bib_ref] Disease resistance mechanisms in plants, Andersen [/bib_ref]. Some successful pathogens can overcome PTI by delivering effectors into plant cells that suppress PTI, thus leading to disease susceptibility. In turn, some plants have evolved another immune response in which microbial effectors (or host proteins modified by effectors) are recognized by intracellular receptor proteins encoded by resistance genes [bib_ref] Origin and evolution of the plant immune system, Han [/bib_ref]. This recognition triggers a rapid and robust defense response, the so-called effector-triggered immunity (ETI) . ETI is often accompanied by a hypersensitive response (HR) at the infection site, a form of programmed cell death [bib_ref] Hypersensitive responses in plants, Thakur [/bib_ref]. However, some PAMPs (e.g., the bacterial Harpin HrpZ protein) can also induce HR in plants [bib_ref] Defence triggered by Flg22 and harpin is integrated into a different stilbene..., Chang [/bib_ref]. Among ROS, H 2 O 2 is relatively stable and is an important molecule regulating plant immunity [bib_ref] Reactive oxygen species signaling in response to pathogens, Torres [/bib_ref]. H 2 O 2 might have a direct antimicrobial role against the invading pathogen and also provokes crosslinking of cell wall components to arrest pathogen invasion. ROS also function as molecules for the activation of defense mechanisms, and triggers localized cell death around the infection site [bib_ref] Arabidopsis gp91phox homologues Atrbohd and Atrbohf are required for accumulation of reactive..., Torres [/bib_ref]. Phytohormones, together with ROS, provide important signals to help orchestrate plant responses to abiotic and biotic stresses. Immune responses are largely coordinated by the phytohormones salicylic acid (SA), ethylene (ET), and jasmonic acid (JA). Plant hormones do not function independently, but synergistic or antagonistic interactions between hormone pathways ultimately drive the fine-tuning of plant defense responses. At the organismal level, interactions between hormone signaling pathways might also balance trade-offs between conflicting biotic and abiotic stress responses (i.e., prioritization of responses in leaves of Arabidopsis plants) . Sugar and ROS have also been proposed as candidate signaling molecules to regulate prioritization between biotic and abiotic stress responses . Plant microRNAs (miRNAs) are a class of small RNA molecules that mediate post-transcriptional gene silencing through sequence complementarity with cognate target transcripts [bib_ref] MicroRNAs: genomics, biogenesis, mechanism, and function, Bartel [/bib_ref] [bib_ref] Expression of Arabidopsis MIRNA genes, Xie [/bib_ref]. They are transcribed from MIR genes as long precursor transcripts that adopt a stem-loop structure by self-complementarity that are processed by a RNase III DICER-like, typically DCL1, to produce a double stranded duplex, the miRNA-5p/miRNA-3p duplex (previously named as miRNA/miRNA*) [bib_ref] The expanding world of small RNAs in plants, Borges [/bib_ref]. The functional strand of the duplex is selectively loaded into an ARGONAUTE protein, the effector protein of the RNA-induced silencing complex (RISC), which mediates post-transcriptional gene silencing by cleaving the target transcripts or by translational inhibition [bib_ref] Widespread translational inhibition by plant miRNAs and siRNAs, Brodersen [/bib_ref] [bib_ref] Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA, Llave [/bib_ref]. The important role of plant miRNAs in diverse developmental processes and adaptation to environmental stresses is well documented [bib_ref] Small RNAs and their roles in plant development, Chen [/bib_ref] [bib_ref] Contribution of small RNA pathway components in plant immunity, Seo [/bib_ref] [bib_ref] MicroRNAs and their regulatory roles in plant-environment interactions, Song [/bib_ref] [bib_ref] Emerging role for RNAbased regulation in plant immunity, Staiger [/bib_ref]. The first plant miRNA demonstrated to be involved in immunity was the Arabidopsis miR393. Here, perception of the elicitor flg22 induces miR393 accumulation and downregulation of auxin receptors, resulting in resistance to bacterial pathogens [bib_ref] A plant miRNA contributes to antibacterial resistance by repressing auxin signaling, Navarro [/bib_ref]. Since then, other miRNAs controlling diverse processes have been shown to be involved in Arabidopsis immunity, either ETI or PTI [bib_ref] Cloning and characterization of small RNAs from Medicago truncatula reveals four novel..., Jagadeeswaran [/bib_ref] [bib_ref] Contribution of small RNA pathway components in plant immunity, Seo [/bib_ref] [bib_ref] A microRNA superfamily regulates nucleotide binding site-leucine-rich repeats and other mRNAs, Shivaprasad [/bib_ref] [bib_ref] Emerging role for RNAbased regulation in plant immunity, Staiger [/bib_ref]. Some examples are miR160, miR396, miR398, miR400, miR472, miR773, miR844, miR858, miR863, and miR156 [bib_ref] The Arabidopsis miR472-RDR6 silencing pathway modulates PAMP-and effector-triggered immunity through the posttranscriptional..., Boccara [/bib_ref] [bib_ref] MiR858-mediated regulation of flavonoid-specific MYB transcription factor genes controls resistance to pathogen..., Camargo-Ram Irez [/bib_ref] [bib_ref] MicroRNA844-guided downregulation of Cytidinephosphate Diacylglycerol Synthase3 (CDS3) mRNA affects the response of..., Lee [/bib_ref] [bib_ref] Identification of microRNAs involved in pathogen-associated molecular patterntriggered plant innate immunity, Li [/bib_ref] [bib_ref] MiRNA863-3p sequentially targets negative immune regulator ARLPKs and positive regulator SERRATE upon..., Niu [/bib_ref] [bib_ref] MicroRNA400-guided cleavage of pentatricopeptide repeat protein mRNAs renders Arabidopsis thaliana more susceptible..., Pandey [/bib_ref] [bib_ref] The microrna miR773 is involved in the arabidopsis immune response to fungal..., Salvador-Guirao [/bib_ref] [bib_ref] MiR156/ SPL9 regulates reactive oxygen species accumulation and immune response in Arabidopsis..., Yin [/bib_ref]. Depending on their target gene, these miRNAs can function as positive or negative regulators in fine-tuning immune responses. Even though pathogen infection has been shown to induce alterations in the expression of a plethora of Arabidopsis miRNAs, the mechanistic role of these miRNAs in processes underling immunity is often unclear. Additionally, most studies on miRNAs involved in plant immunity have been carried out in the interaction of Arabidopsis plants with the bacterial pathogen Pseudomonas syringae, and less is known on miRNAs involved in resistance against fungal pathogens. On the other hand, distinct miRNAs have been associated with regulation of nutrient homeostasis in plants . Perhaps the best-known example is miR399, which is involved in the control of phosphate (Pi) homeostasis in Arabidopsis plants . Under limiting Pi conditions, miR399 accumulation increases and causes repression of its target gene, PHOSPHATE2 (PHO2), encoding an E2 ubiquitin-conjugating enzyme responsible for degradation of phosphate transporters [bib_ref] A miRNA involved in phosphate-starvation response in Arabidopsis, Fujii [/bib_ref] [bib_ref] Identification of downstream components of ubiquitinconjugating enzyme PHOSPHATE2 by quantitative membrane proteomics..., Huang [/bib_ref] [bib_ref] -dependent degradation of PHO1 modulates phosphate homeostasis in Arabidopsis, Liu [/bib_ref]. Hence, miR399 accumulation in response to Pi starvation relieves negative posttranscriptional control of Pi transporters and promotes uptake of Pi in Arabidopsis plants. The miR399/PHO2 regulatory module has been also recognized as a regulator of Pi homeostasis in rice (Oryza sativa) [bib_ref] MicroRNA-mediated signaling and regulation of nutrient transport and utilization, Chien [/bib_ref] [bib_ref] Novel signals in the regulation of Pi starvation responses in plants: facts..., Puga [/bib_ref]. In this work, we investigated whether miR399 plays a role in disease resistance in Arabidopsis. We show that miR399 overexpression and loss-of-function of PHO2 causes an increase in Pi levels, these plants also exhibiting resistance to infection by necrotrophic (Plectosphaerella cucumerina) and hemibiotrophic (Colletotrichum higginsianum) fungal pathogens. Growing Arabidopsis plants under high Pi supply also increases resistance to infection by these pathogens. In the absence of pathogen infection, plants that overaccumulate Pi (e.g., miR399 overexpressors, pho2 mutants, and wild-type plants grown under high Pi supply) showed ROS accumulation, increased SA and JA levels, and upregulation of genes involved in SAand JA-dependent defense pathways. Pathogen infection was found to be associated with a higher production of ROS in high Pi plants. Of note, during pathogen infection, induction of the ERF1 branch of the JA pathway and repression of the MYC2/VSP2 branch occur in high Pi plants. Overall, our results support the notion that an increase in Pi content has an impact on hormone networks regulating Arabidopsis defense and promotes resistance to pathogen infection in Arabidopsis. These results are markedly different from those recently reported in rice , where miR399 overexpression and high Pi supply were found to enhance susceptibility to infection by the rice blast fungus Magnaporthe oryzae. These findings illustrate the need of investigating the effects of nutrient supply on the expression of immune responses and disease resistance on a case-by-case basis. # Results ## Resistance to infection by fungal pathogens in arabidopsis plants overexpressing mir399 In Arabidopsis, the miR399 family comprises six members, MIR399a-f. Of these, miR399f has identical mature sequences in Arabidopsis and rice. Previous studies indicated that transgenic Arabidopsis and rice plants overexpressing miR399f overaccumulate Pi in leaves [bib_ref] Regulation of phosphate homeostasis by MicroRNA in Arabidopsis, Chiou [/bib_ref]. In this work, we investigated whether miR399f overexpression, and subsequent Pi accumulation, has an effect on resistance to pathogen infection in Arabidopsis. To this end, transgenic plants overexpressing MIR399f (hereinafter referred to as miR399 OE plants) were generated. Compared to wild-type plants, miR399 OE lines accumulated precursor and mature miR399 sequences. The observed increase in miR399 abundance in miR399 OE lines was accompanied by a decrease in PHO2 transcript level and overaccumulation of Pi in rosette leaves [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref]. The miR399 OE lines were challenged with the fungus P. cucumerina, the causal agent of the sudden death and blight disease in many dicotyledonous species. The Arabidopsis-P. cucumerina pathosystem is a widely used model system for studies on disease resistance against necrotrophic fungi (S anchez- [bib_ref] Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic..., Vallet [/bib_ref] [bib_ref] b-amino-butyric acid-induced resistance against necrotrophic pathogens is based on ABA-dependent priming for..., Ton [/bib_ref]. At the time of pathogen inoculation (3week-old plants), no obvious phenotypic differences were observed between miR399 OE plants and wild-type plants [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref]. However, at a later developmental stage, the miR399 and pho2 plants displayed symptoms of Pi excess (e.g., chlorosis on mature leaves of adult plants), most probably, because of Pi accumulation overtime (results not shown; similar results were previously reported by [bib_ref] Regulation of phosphate homeostasis by MicroRNA in Arabidopsis, Chiou [/bib_ref]. Upon pathogen challenge, wild-type plants were severely affected by P. cucumerina, while the miR399 OE plants consistently exhibited enhanced resistance . While 75-85% of miR399 OE plants survived at 7 dpi, only 20% of the wild-type plants were able to overcome infection [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref] , left panel). Quantitative PCR (qPCR) measurement of fungal DNA confirmed that less fungal biomass was present in leaves of miR399 OE plants compared with leaves of wild-type plants infected with P. cucumerina [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref] , right panel), which is consistent with the phenotype of resistance that is observed in miR399 OE plants. Trypan blue staining was used to visualize both fungal structures and dead cells in the fungal-infected leaves of wild-type and miR399 OE plants. Whereas extensive fungal growth occurred in leaves of wild-type plants, no fungal growth could be observed in leaves of miR399 OE plants [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref]. Instead, scattered groups of dead cells were visualized in P. cucumerina-infected miR399 OE plants [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref] , right panels). Since P. cucumerina is a necrotrophic fungus, we hypothesized that the effect of miR399 overexpression on disease resistance might be dependent on the lifestyle of this pathogen. Accordingly, we investigated resistance of miR399 OE plants to infection by the hemibiotrophic fungus C. higginsianum. This fungus causes the anthracnose leaf spot disease on Brassica species, as well as A. thaliana [bib_ref] A novel Arabidopsis-Colletotrichum pathosystem for the molecular dissection of plant-fungal interactions, O&apos;connell [/bib_ref]. The miR399 OE plants showed resistance to C. higginsianum infection relative to wild- Collectively, these results demonstrate that miR399 overexpression enhances resistance to infection by necrotrophic (P. cucumerina) and hemibiotrophic (C. higginsianum) fungal pathogens. Hence, it is likely that miR399-mediated resistance in Arabidopsis does not depend on the lifestyle of the fungus. A pattern of cell death occurs in the fungal-infected miR399 OE plants. pho2 mutant plants exhibit resistance to infection by fungal pathogens miR399 targets and suppresses the expression of PHO2, encoding the ubiquitin-conjugating enzyme that mediates the degradation of Pi transporter proteins [bib_ref] A miRNA involved in phosphate-starvation response in Arabidopsis, Fujii [/bib_ref] [bib_ref] Identification of downstream components of ubiquitinconjugating enzyme PHOSPHATE2 by quantitative membrane proteomics..., Huang [/bib_ref]. A loss-offunction allele of PHO2 was previously described [bib_ref] Characterization of a phosphateaccumulator mutant of Arabidopsis thaliana, Delhaize [/bib_ref]. This mutant allele possesses a single nucleotide mutation that causes premature termination and loss of ubiquitin conjugation activity of PHO2 [fig_ref] Figure 2: Resistance of miR399 OE plants to infection by the hemibiotrophic fungus C [/fig_ref]. pho2 plants resemble miR399 OE plants in that they both show Pi overaccumulation in leaves resulting from increased Pi uptake from roots and root-to-shoot translocation [fig_ref] Figure 2: Resistance of miR399 OE plants to infection by the hemibiotrophic fungus C [/fig_ref]. We therefore hypothesized that loss-of-function of PHO2 might result in a similar disease phenotype as suppression of PHO2 by overexpression of miR399. The pho2 plants were then examined for pathogen resistance. No phenotypic differences were observed between pho2 and wild-type plants at the time of inoculation . Consistent with the disease phenotype observed in miR399 OE plants, the pho2 mutant exhibited resistance to infection by P. cucumerina [fig_ref] Figure 3: Resistance to infection by fungal pathogens in pho2 plants [/fig_ref] , which was confirmed by quantifying the survival ratio of the infected plants and the amount of fungal biomass [fig_ref] Figure 3: Resistance to infection by fungal pathogens in pho2 plants [/fig_ref]. Trypan blue staining revealed a pattern of dead cells in leaves of pho2 mutants that have been inoculated with P. cucumerina spores [fig_ref] Figure 3: Resistance to infection by fungal pathogens in pho2 plants [/fig_ref] , as was also observed in miR399 OE plants. Finally, the pho2 plants also showed resistance to C. higginsianum infection as compared with wild-type plants [fig_ref] Figure 3: Resistance to infection by fungal pathogens in pho2 plants [/fig_ref]. Taken together, results here presented support the notion that both pho2 and miR399 OE plants accumulate Pi in leaves and exhibit resistance to infection by fungal pathogens with a necrotrophic or hemibiotrophic lifestyle. Homozygous miR399 OE lines (#1, #4, and #10) and wild-type plants were grown in soil for 3 weeks and then assayed for disease resistance. Three independent experiments were carried out with at least 12 plants per line in each experiment. (a) Accumulation of precursor (pre-miR399) and mature miR399 sequences was determined by RT-qPCR and stemloop RT-qPCR, respectively. Expression of the miR399 target AtPHO2 was determined by RT-qPCR. The Arabidopsis b-tubulin2 gene (At5g05620) was used to normalize transcript levels (relative expression). The accumulation of free Pi in leaves is shown (right panel). Bars represent mean AE SEM of three biological replicates with at least three plants per replicate (t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). (b) Plants were spray-inoculated with P. cucumerina spores (5 9 10 5 spores ml À1 ). Pictures were taken at 7 days post-inoculation (dpi ## Mir399 expression is upregulated during fungal infection and treatment with fungal elicitors To gather further support for the involvement of miR399 in Arabidopsis immunity, we investigated whether fungal infection or treatment with fungal elicitors provokes alterations in MIR399 expression in wild-type plants. The accumulation of miR399f precursor transcripts was examined by reverse transcription qPCR (RT-qPCR), while mature miR399 abundance was determined by stem-loop RT-qPCR. Differences among mature miR399 sequences are found at their 3 0 terminal region (miRBase, https://www. mirbase.org/). This fact allowed us to design miR399fspecific primers for stem-loop RT-qPCR analysis. The PCRamplified products were further confirmed through DNA sequencing. Fungal-responsiveness of MIR399f was examined at 48 and 72 h post-inoculation (hpi) with P. cucumerina spores. An increase in the accumulation of both precursor and mature miR399 sequences was clearly observed in the response of wild-type plants to pathogen infection. When examining the PHO2 expression behavior in response to infection, an opposite profile was observed between MIR399f and PHO2 at the earliest time of infection here assayed (48 hpi with P. cucumerina spores). At 72 hpi, however, the accumulation of PHO2 transcripts in fungal-infected wild-type plants was similar to that in mock-inoculated plants. In PTI, the activation of defense mechanisms relies on the detection of PAMPs (commonly referred to as elicitors). Accordingly, we investigated whether miR399 accumulation is affected by treatment with a crude preparation of Leaves were locally inoculated with a spore suspension at 4 9 10 6 spores ml À1 . Results are from one out of three independent experiments performed with three independent miR399 OE lines (lines 1, 4, and 10) and wild-type plants which gave similar results. At least 12 plants per genotype were assayed in each experiment. (a) Disease symptoms at 7 days post-inoculation (dpi) with fungal spores. The diseased leaf area was quantified using ImageJ software. Quantification of C. higginsianum DNA was carried out by qPCR using specific primers for the C. higginsianum Internally transcribed spacer 2 Three independent experiments were carried out with similar results with at least 12 plants per genotype. (a) Disease phenotype of wild-type and pho2 plants upon inoculation with P. cucumerina spores (5 9 10 5 spores ml À1 ). Pictures were taken at 7 days post-inoculation (dpi). (b) Survival ratio of wild-type and pho2 plants at 7 dpi (left panel). Quantification of P. cucumerina DNA was carried out using specific primers for P. cucumerina btubulin at 7 dpi (right panel elicitors obtained by autoclaving and sonicating P. cucumerina mycelium. Similar to P. cucumerina infection, elicitor treatment induced the accumulation of both miR399 precursor and mature sequences. Furthermore, an opposite profile was observed between MIR399 and PHO2 expression at 90 min of elicitor treatment (e.g., upregulation of MIR399f and downregulation of PHO2), but this anticorrelation in expression patterns was not observed at 120 min of elicitor treatment. The observation that pathogen infection and treatment with fungal elicitors are accompanied by upregulation of MIR399 expression suggests that miR399 might function in PTI. Taking into account that miR399 has been shown to negatively regulate PHO2 via the canonical mechanism of cleavage of PHO2 transcripts, results here presented point to the existence of regulatory mechanisms for the control of PHO2 expression other than miR399-guided cleavage of PHO2 transcripts during pathogen infection or treatment with fungal elicitor (as inferred from results obtained at 72 hpi with P. cucumerina spores or 120 min of elicitor treatment). Supporting this possibility, it was recently reported that PHO2 might be modulated by regulatory mechanisms independent of miR399-directed regulation in the response of Arabidopsis plants to salt stress [bib_ref] Molecular manipulation of the miR399/PHO2 expression module alters the salt stress response..., Pegler [/bib_ref]. In that study, no anticorrelation between miR399 accumulation and PHO2 transcript abundance was reported. Whether additional factors are involved in the regulation of PHO2 during pathogen infection deserves further investigation. ## Stimulation of ros production in arabidopsis plants containing increased levels of pi One of the hallmarks of host-pathogen interactions is the overproduction of ROS as a plant defense mechanism, the so-called oxidative burst. ROS include various forms of reactive molecules, such as superoxide radicals (O 2 .À ), hydroxyl radicals (OH . ), and hydrogen peroxide (H 2 O 2 ). Of these, H 2 O 2 might act as both signaling molecule for the activation of plant immune responses and antimicrobial agent [bib_ref] Reactive oxygen species signaling in response to pathogens, Torres [/bib_ref]. Respiratory Burst Oxidase Homolog D (RBOHD), a member of the Arabidopsis Nicotinamide Adenine Dinucleotide Phosphate (NADPH) oxidase gene family, has been shown to be responsible for ROS production after pathogen infection [bib_ref] Regulation of the NADPH oxidase RBOHD during plant immunity, Kadota [/bib_ref] [bib_ref] Arabidopsis gp91phox homologues Atrbohd and Atrbohf are required for accumulation of reactive..., Torres [/bib_ref]. Furthermore, ROS may promote cell death and limit pathogen spread. . Acumulation of precursor (pre-miR399), mature (miR399), and PHO2 transcripts in wild-type plants in response to inoculation with P. cucumerina spores or treatment with P. cucumerina elicitors. The accumulation of pre-miR399f and mature miR399f sequences was determined by RT-qPCR and stem-loop RT-qPCR, respectively, at the indicated times after inoculation with fungal spores (a) or elicitor treatment (b). At 48 and 72 hpi with P. cucumerina spores, pre-miR399 levels increased 4.5-and 6.0-fold, respectively. At the same time points, miR399f levels increased 2.0-and 9.0-fold. Elicitor treatment elevated the level of pre-miR399f accumulation 5.0-and 267-fold at 90 and 120 min of treatment, respectively. MiR399f levels increased 20-and 147-fold in response to elicitor treatment (90 and 120 min, respectively). Four biological replicates and three technical replicates per time point were assayed. Statistically significant differences were determined by ANOVA followed by Tukey's HSD test, where different letters represent statistically significant differences. Ó 2022 The Authors. Knowing that miR399 OE and pho2 plants exhibit a pattern of cell death upon pathogen infection, we examined ROS accumulation in miR399 OE and pho2 plants, both in the presence and in the absence of pathogen infection. Histochemical detection of H 2 O 2 was carried out using the fluorescent probe 2',7'-dichlorofluorescein diacetate (H 2 DCFDA). H 2 DCFDA was previously shown to detect different forms of ROS, mainly H 2 O 2 but also hydroxyl radicals and superoxide anions [bib_ref] Whole-plant live imaging of reactive oxygen species, Fichman [/bib_ref]. Compared with wild-type plants, a higher level of ROS accumulation could be observed in leaves of miR399 OE and pho2 plants compared to wild-type plants in the absence of pathogen infection , upper panels). Plectosphaerella cucumerina infection further increased ROS levels in miR399 OE and pho2 plants , lower panels). Discrete regions accumulating ROS, most probably, correspond to infection sites. Similar results were observed by 3,3'-diaminobenzidine (DAB) staining of P. cucumerina-infected leaves [fig_ref] Figure 3: Resistance to infection by fungal pathogens in pho2 plants [/fig_ref]. Moreover, H 2 DCFDA staining revealed a higher level of ROS accumulation in high Pi plants (e.g., miR399 OE and pho2 plants) that have been treated with P. cucumerina elicitors compared with elicitor-treated wild-type plants. Additionally, we examined ROS generated in miR399 OE, pho2, and wild-type plants in response to treatment with P. cucumerina elicitors using the luminol method. This study confirmed a higher production of ROS after elicitor treatment in leaves of miR399 OE and pho2 plants compared to that in wild-type plants . Collectively, these findings support ROS accumulation in leaves of miR399 OE and pho2 plants in the absence of pathogen infection. During infection with P. cucumerina or treatment with elicitors obtained from this fungus, miR399 OE and pho2 plants produced higher levels of ROS than wild-type plants. In concordance with results obtained by histochemical detection of ROS and measurement of ROS production using the luminol assay, the miR399 OE and pho2 plants exhibited upregulation of RBOHD in the absence of pathogen infection compared with wild-type plants , black bars). Plectosphaerella cucumerina infection further induced RBOHD expression in all the genotypes (wild-type, miR399 OE, and pho2 plants), but its expression reached higher levels in miR399 OE and pho2 plants than in wildtype plants . Then, ROS accumulation in miR399 OE and pho2 plants can be explained, at least partially, by an increased RBOHD expression. However, other factors causing an increase in ROS accumulation cannot be discarded. The observation that both miR399 OE and pho2 plants accumulated Pi and ROS, these plants also exhibiting enhanced disease resistance [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref] , prompted us to investigate whether ROS production and disease resistance are affected by Pi supply in Arabidopsis. To address this question, wild-type plants were grown in vitro on media at different Pi concentrations (0.05, 0.1, and 0.25 mM Pi, hereinafter referred to as P 0.05 , P 0.1 , and P 0.25 plants). As expected, measurement of Pi content confirmed that increasing Pi supply to wild-type plants results in higher leaf Pi content . Most importantly, increasing Pi supply was accompanied by an increase in ROS accumulation as revealed by H 2 DCFDA staining of leaves from Pi-treated plants . Similarly, luminol assays demonstrated that treatment with Pi fosters ROS production in wild-type plants, with ROS production correlating well with Pi concentration . Upon pathogen inoculation, high Pi plants consistently displayed enhanced resistance to infection by either P. cucumerina or C. higginsianum ,f, respectively). Pi-induced resistance to pathogen infection in Arabidopsis is associated with modulation of the SA-and JAdependent defense pathways As previously mentioned, SA and JA play a critical role in the transcriptional reprogramming of Arabidopsis plants in . Enhanced accumulation of ROS and disease resistance in Arabidopsis plants that overaccumulate Pi. (a) In situ histochemical detection of ROS in leaves of miR399 OE and pho2 plants. Plants were spray-inoculated with P. cucumerina spores (5 9 10 5 spores ml À1 ) or mock-inoculated. Visualization of H 2 O 2 accumulation was carried out using the fluorescent probe H 2 DCFDA at 2 days post-inoculation (dpi). Bars represent 200 lm. (b) Expression of RBOHD in mockinoculated and P. cucumerina-inoculated plants at 24 hpi (black and gray bars, respectively). The expression values were normalized to the Arabidopsis b-tubulin2 gene (At5g62690). Three biological replicates (with three plants per replicate) were examined. Different letters represent statistically significant differences (ANOVA followed by Tukey's HSD test; P < 0.05). (c) Free Pi content in plants that have been grown under different conditions of Pi supply. Plants were grown on agar plates for 1 week and transferred to fresh agar plates with medium containing different concentrations of Pi (0.05, 0.1, or 0.25 mM). Plants were allowed to continue growth for one more week and then inoculated with fungal spores. Pi content was determined at the time of inoculation with fungal spores. (d) ROS accumulation in wild-type Arabidopsis (Col-0) plants that have been grown under different Pi supply conditions, that is, 0.05, 0.1, 0.25, or 2 mM Pi (P 0.05 , P 0.1 , P 0.25 , and P 2 , respectively). ROS were detected using H 2 DCFDA. Representative images are shown. Bars correspond to 1 mm. (e) Resistance to infection by P. cucumerina in Pi-treated Arabidopsis plants. Appearance of plants at 7 dpi) with P. cucumerina spores (4 9 10 6 spores ml À1 ; left panel). Representative results from one of three independent infection experiments that gave similar results are shown. Right panel, fungal biomass determined at 3 dpi by qPCR analysis using specific primers for P. cucumerina b-tubulin and normalized to the Arabidopsis UBIQUITIN21 gene (At5g25760). (f) Resistance of Pi-treated Arabidopsis plants to infection by C. higginsianum. Disease symptoms of Arabidopsis plants at 12 dpi with C. higginsianum spores (5 9 10 5 spores ml À1 ). Right panel, fungal biomass at 7 dpi as determined by qPCR analysis using specific primers for C. higginsianum Internally transcribed spacer 2 (ITS2). Means of three biological replicates, each one from a pool of at least three plants, are shown in (e) and (f) (right panels; t-test, *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001). response to pathogen infection [bib_ref] Hormonal modulation of plant immunity, Pieterse [/bib_ref]. A pathogen-induced accumulation of ROS is also required for the induction of SA-dependent defenses, indicating that ROS and SA are intertwined in a complex regulatory network (Herrera-V asquez et al., 2015; [bib_ref] Free radicals mediate systemic acquired resistance, Wang [/bib_ref]. To get deeper insights into the mechanisms underlying disease resistance in miR399 OE and pho2 plants, we investigated the expression of defense genes linked to the SA and JA pathways in these plants. The marker genes of the SA-mediated defense response here examined were: Pathogenesis-Related 1 (PR1), Non-expressor of Pathogenesis-Related genes 1 (NPR1), and Phytoalexin Deficient4 (PAD4) [bib_ref] Arabidopsis thaliana PAD4 encodes a lipase-like gene that is important for salicylic..., Jirage [/bib_ref]. We found that in the absence of pathogen infection, PR1, NPR1, and PAD4 expression was upregulated in both miR399 OE and pho2 plants compared with wild-type plants , black bars). PR1, NPR1, and PAD4 expression further increased in response to P. cucumerina infection in miR399 OE and pho2 plants, but not in wild-type plants , gray bars). Regarding the JA pathway, two branches are documented in Arabidopsis: the MYC2 branch, which is regulated by AtMYC2 (a basic helix-loop-helix-leucine zipper transcription factor), and the ERF branch, which is regulated by AtERF1 (a member of the APETALA/ERF transcription factor family) [bib_ref] JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated..., Lorenzo [/bib_ref]. Plant Defensin 1.2 (PDF1.2) is commonly used as marker of the ERF branch, whereas the MYC branch is marked by the induction of Vegetative Storage Protein 2 (VSP2) [bib_ref] JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated..., Lorenzo [/bib_ref] [bib_ref] Hormonal modulation of plant immunity, Pieterse [/bib_ref] [bib_ref] Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth..., Wasternack [/bib_ref] [bib_ref] Jasmonate signaling and manipulation by pathogens and insects, Zhang [/bib_ref]. The ERF branch and the MYC branch of the JA signaling pathway have been reported to repress each other [bib_ref] JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated..., Lorenzo [/bib_ref] [bib_ref] Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth..., Wasternack [/bib_ref]. Compared with wild-type plants, the miR399 OE and pho2 plants showed higher expression of transcription factor and defense marker genes associated to the ERF1/ PDF1.2 and MYC2/VSP2 branches of the JA signaling pathway in the absence of pathogen infection ,c, black bars). Pathogen infection further induced ERF1/PDF1 expression in wild-type, miR399 OE, and pho2 plants , gray bars). Of note, while MYC2/VSP2 expression was not significantly affected by pathogen infection in wild-type plants, their expression was found to be repressed in miR399 OE plants. Pathogen infection downregulated VSP2 expression in pho2 plants, but not in wildtype plants . Together, these findings support the notion that resistance to pathogen infection in plants accumulating Pi (i.e., miR399 OE and pho2 plants) is associated with a higher expression of SA-and JAregulated genes under non-infection conditions. A pathogen-induced superactivation of genes involved in the SA pathway and the ERF1 branch of the JA pathway occurs in these plants, while pathogen infection represses the MYC2 branch of the JA signaling pathway. Further, we measured the levels of the phytohormones SA and JA in miR399 OE and pho2 plants. Compared with wild-type plants, miR399 OE and pho2 plants accumulated significantly higher levels of SA under normal growth conditions (e.g., in the absence of pathogen infection) . This observation is consistent with the expression pattern of SA-responsive defense genes found in miR399 OE and pho2 plants (see . Upon pathogen infection, however, there were no significant differences in SA levels among wild-type, miR399 OE, and pho2 plants , upper left panel, gray bars). We also noticed that SA glucoside (SAG; the storage form of SA) accumulated at substantially higher levels in the fungal-infected miR399 OE and pho2 plants compared with the fungal-infected wild-type plants . These observations points to a strict control of the SA level in miR399 OE and pho2 plants. In the absence of pathogen infection, miR399 OE and pho2 plants accumulated more JA than wild-type plants , lower left panel, black bars). JA content further increased during pathogen infection in wild-type and miR399 OE plants , lower left panel, gray bars). The level of 12-oxophytodienoic acid (OPDA), a JA precursor, in miR399 OE and pho2 plants did not differ significantly from that in wild-type plants . Additionally, we investigated whether Pi treatment modulates SA and JA signaling pathways. Increasing Pi supply results in a gradual increase in SA and JA levels (as well as SAG and OPDA levels) . These results are in concordance with the upregulation of PR1, NPR1, and PDF1.2 in response to Pi treatment . Collectively, these results support the notion that in the absence of pathogen infection, miR399 overexpression, loss-of-function of PHO2, and Pi treatment result in higher levels of SA and JA and subsequent upregulation of SAand JA-dependent defense gene expression. A Pi-mediated modulation of the SA and JA signaling pathways may contribute to the phenotype of disease resistance that is observed in Arabidopsis plants overaccumulating Pi, namely miR399 OE, pho2, and wild-type plants grown under high Pi supply. # Discussion Nutrients play crucial roles in normal plant growth and development, and nutrient imbalance might also have a substantial impact on the predisposition of plants to resist pathogen attack. Depending on the identity of the interacting partners, nutritional imbalances caused by either nutrient excess or deficiency may determine the outcome of the interaction, resistance or susceptibility [bib_ref] Fertilization affects severity of disease caused by fungal plant pathogens, Veresoglou [/bib_ref]. On the other hand, although miRNA-mediated regulation of gene expression in processes involved in either nutrient stress or immune responses is well documented, less effort has been made to investigate miRNA In this study, we provide evidence that increasing Pi content in Arabidopsis results in enhanced disease resistance. Several pieces of evidence support this conclusion. On the one hand, we show that Pi accumulation caused by miR399 overexpression, loss-of-function of PHO2, or treatment with Pi confers resistance to infection by fungal expression might be indicative that miR399 functions in PTI. Altogether, these findings support the notion that miR399 overexpression has a positive impact on resistance to pathogen infection in Arabidopsis and reinforce the notion that miR399 plays a dual role in plants by controlling Pi homeostasis and disease resistance. Another finding of this study is that, under non-infection conditions, overaccumulation of Pi in Arabidopsis leaves is accompanied by production of ROS, their levels increasing significantly during pathogen infection. Hence, ROS accumulation in miR399 OE and pho2 plants might be responsible for the pattern of cell death observed in these plants during pathogen infection, a response that is reminiscent of the pathogen-induced HR. In contrast, the fungalinfected wild-type plants did not exhibit cell death and showed extensive fungal growth. Additionally, elevated levels of ROS might contribute to the activation of immune responses in high Pi plants leading to a phenotype of disease resistance. From this point of view, it might be interesting to determine whether Pi-induced ROS accumulation can be generalized to other plant species. At the time of inoculation with fungal spores (3-week-old plants), miR399 OE and pho2 plants accumulated 3-4 times more Pi than wild-type plants. By this developmental stage, plant growth is not compromised in plants accumulating this level of Pi. It is tempting to hypothesize that this increase in Pi content and/or ROS levels is perceived by the host plant as a stressful situation and that the plant responds to these signals with the induction of defense gene expression. An increase in Pi content might eventually increase intracellular and extracellular Pi levels, increasing ATP levels. Here, it should be mentioned that ATP has been shown to function as a DAMP signal after release into the extracellular space upon cellular damage and that extracellular ATP enhances plant defense against pathogens through the activation of JA signaling [bib_ref] Extracellular ATP acts as a damage-associated molecular pattern (DAMP) signal in plants, Tanaka [/bib_ref] [bib_ref] Extracellular ATP acts on jasmonate signaling to reinforce plant defense, Tripathi [/bib_ref]. Further studies are needed to establish whether ATP levels are altered in high Pi plants. Gene expression analysis revealed regulation of the SA and JA defense signaling pathways in Arabidopsis plants overaccumulating Pi in leaves. Thus, high Pi plants (miR399 OE and pho2 plants) showed activation of SAregulated and JA-regulated genes under non-infection conditions. SA and JA were reported to play a positive role in the regulation of resistance to P. cucumerina infection in Arabidopsis [bib_ref] Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi, Berrocal-Lobo [/bib_ref] [bib_ref] Disruption of abscisic acid signaling constitutively activates Arabidopsis resistance to the necrotrophic..., Vallet [/bib_ref]. Previous studies also revealed that PDF1.2 induction was associated to resistance to infection by necrotrophic fungi, including P. cucumerina, in Arabidopsis [bib_ref] Constitutive expression of ETHYLENE-RESPONSE-FACTOR1 in Arabidopsis confers resistance to several necrotrophic fungi, Berrocal-Lobo [/bib_ref]. Consistent with the upregulation of genes involved in the SA and JA signaling pathways, SA and JA accumulated in non-infected high Pi plants. In other studies, a feed-forward loop between SA and ROS production (e.g., H 2 O 2 ) has been reported in which ROS are involved both upstream and downstream of SA in the plant defense response to pathogen infection [bib_ref] Salicylic acid and reactive oxygen species interplay in the transcriptional control of..., Herrera-V Asquez [/bib_ref]. We hypothesize that Pi content and possibly also ROS accumulation might influence defense hormone signaling. Interestingly, a different regulation in the two branches of the JA pathway was observed in high Pi plants during pathogen infection. Whereas ERF1 and PDF1.2 expression is further increased during infection in miR399 OE and pho2 plants (infected versus non-infected plants), VSP2 expression diminished in miR399 OE and pho2 plants (infected versus non-infected plants). We envisage that this differential regulation might be due to still unknown factors that cooperate in an antagonistic manner in the regulation of PDF1.2 and VSP2 expression during infection with the fungal pathogen P. cucumerina. In line with this, a negative correlation between the ERF1 and MYC2 branches has been previously described in the Arabidopsis response to different attackers such as pathogens and phytophagous insects [bib_ref] JASMONATE-INSENSITIVE1 encodes a MYC transcription factor essential to discriminate between different jasmonate-regulated..., Lorenzo [/bib_ref] [bib_ref] Hormonal modulation of plant immunity, Pieterse [/bib_ref] [bib_ref] Jasmonates: biosynthesis, perception, signal transduction and action in plant stress response, growth..., Wasternack [/bib_ref] [bib_ref] Jasmonate signaling and manipulation by pathogens and insects, Zhang [/bib_ref]. Here, necrotrophic pathogens preferentially activate the ERF branch, while the MYC2 branch is activated by insect herbivory and wounding. A specialization in the host plant for modulation of each branch of the JA signaling pathway might exist. It will be of interest to explore whether Pi accumulation has an effect on plant-insect interactions in Arabidopsis. Clearly, interactions between defense-related hormone pathways provide the plant with a powerful regulatory potential to control defense responses [bib_ref] Coronatine promotes pseudomonas syringae virulence in plants by activating a signaling cascade..., Zheng [/bib_ref]. However, the type of induced response that is effective for disease resistance appears to vary depending on the host plant and pathogen identity. Although there are exceptions, pathogens with a biotrophic or hemibiotrophic lifestyle (such as P. syringae) are generally more sensitive to SA-dependent responses, whereas necrotrophic pathogens are commonly deterred by JA/ET-dependent defenses [bib_ref] Contrasting mechanisms of defense against biotrophic and necrotrophic pathogens, Glazebrook [/bib_ref]. The observation that high Pi plants (e.g., miR399 OE, pho2, and plants grown under high Pi supply conditions) show enhanced resistance to necrotrophic (P. cucumerina) and hemibiotrophic (C. higginsianum) fungal pathogens makes it unlikely that the pathogen lifestyle determines disease resistance in high Pi plants. Based on the results obtained in this study, a model is proposed to describe possible mechanisms underlying disease resistance in high Pi Arabidopsis plants [fig_ref] Figure 8: Proposed model to explain how Pi content and defense responses are integrated... [/fig_ref]. According to our model, miR399 overexpression and lossof-function of PHO2, as well as growing plants under high Pi supply, increases Pi accumulation, ROS production, and SA and JA accumulation. A higher expression of SA-and JA-dependent defense responses in Arabidopsis plants accumulating Pi would allow the plant to mount a successful defense response upon encountering a pathogen. The existence of links between components of the phosphate starvation response (PSR) and disease resistance has been previously described [bib_ref] The impact of phosphorus on plant immunity, Chan [/bib_ref]. For instance, enhanced resistance to infection by the bacterial pathogen P. syringae pv. tomato DC3000 and the oomycete pathogen Hyaloperonospora arabidopsidis was reported in phr1 mutant plants, PHR1 being the master transcriptional regulator of the Arabidopsis PSR [bib_ref] Root microbiota drive direct integration of phosphate stress and immunity, Castrillo [/bib_ref]. This study also led the authors to propose that PHR1 might fine-tune JA responses under Pi starvation in specific biological contexts, rather than being a regulator of the JA signaling pathway [bib_ref] Root microbiota drive direct integration of phosphate stress and immunity, Castrillo [/bib_ref]. In other studies, transgenic expression of a phytoplasma effector (SAP11) in Arabidopsis was found to trigger PSRs that are mainly dependent on PHR1 [bib_ref] Transgenic plants that express the phytoplasma effector SAP11 show altered phosphate starvation..., Lu [/bib_ref]. The SAP11 transgenic plants overaccumulated Pi in leaves and were more susceptible to P. syringae pv. tomato DC3000 infection [bib_ref] Transgenic plants that express the phytoplasma effector SAP11 show altered phosphate starvation..., Lu [/bib_ref]. The PSR system also appears to control root colonization by the endophytic fungus Colletotrichum tofieldiae in Arabidopsis [bib_ref] A Network of phosphate starvation and immune-related signaling and metabolic pathways controls..., Frerigmann [/bib_ref] [bib_ref] Root endophyte Colletotrichum tofieldiae confers plant fitness benefits that are phosphate status..., Hiruma [/bib_ref]. Very recently, [bib_ref] Direct inhibition of phosphate transport by immune signaling in Arabidopsis, Dindas [/bib_ref] described a negative regulation of Pi transport by immune signaling in Arabidopsis. In citrus plants, Pi content was associated to symptomology in Huanglongbing (HLB) disease, where Pi deficiency favors development of HLB symptoms [bib_ref] Small RNA profiling reveals phosphorus deficiency as a contributing factor in symptom..., Zhao [/bib_ref]. In plant-insect interactions, Pi deficiency was found to induce the JA signaling pathway to enhance resistance to insect herbivory in a process that is partially under the control of PHR1 [bib_ref] Phosphate deficiency induces the jasmonate pathway and enhances resistance to insect herbivory, Khan [/bib_ref]. Collectively, results here presented together with those found in the literature in other pathosystems support the existence of connections between Pi homeostasis and immune signaling. These mechanisms should operate in a coordinated manner to properly balance nutrient responses and plant immunity. Results from our study also raise intriguing questions about the impact of Pi content on disease resistance in different pathosystems. In the present study we show that an increase in Pi content positively regulates Arabidopsis immune responses, which, in turn, enhances resistance to infection by fungal pathogens. Contrarily, in rice plants, a higher Pi content caused by miR399 overexpression or high Pi fertilization was found to negatively regulate defense gene expression, thus increasing susceptibility to infection by the blast fungus M. oryzae . In other studies, Pi deficiency was found to enhance resistance to Verticillium dahliae in cotton (Gossypium hirsutum) [bib_ref] Phosphate deficiency enhances cotton resistance to Verticillium dahliae through activating jasmonic acid..., Luo [/bib_ref] and insect herbivory in Arabidopsis [bib_ref] Phosphate deficiency induces the jasmonate pathway and enhances resistance to insect herbivory, Khan [/bib_ref]. Together, our results and those previously reported support the notion that Pi content might positively or negatively regulate disease resistance depending on the interacting partners. It is tempting to speculate that different plants might have evolved diverse mechanisms to adapt to Pi alterations which, in turn, would determine a different effect of Pi in the regulation of immune responses. It is likely that integration of Pi stress (either deficiency or excess) and immune responses might vary depending on the host plant and the type of pathogen, the outcome of the interaction being also dependent on the role of the defense hormones SA and JA in that particular interaction. Alternatively, Pi might affect growth and/or pathogenicity of a fungal pathogen, either by creating a less favorable environment for pathogen growth or by reducing the production of pathogen virulence factors. Clearly, these aspects deserve further investigation. ## High-pi supply mir399 overexpression / pho2 Pi The interplay between Pi, ROS, and hormones would allow the plant to mount an effective immune response. Although crosstalk between ROS and hormonal pathways has been described [bib_ref] Salicylic acid and reactive oxygen species interplay in the transcriptional control of..., Herrera-V Asquez [/bib_ref] [bib_ref] Interplay between reactive oxygen species and hormones in the control of plant..., Xia [/bib_ref] , further investigation is needed to elucidate (i) the exact mechanisms by which Pi content modulates ROS production and hormone content and (ii) how these signaling pathways communicate with each other in the coordinated regulation of Pi homeostasis and immune responses. ## No infecɵon ## Disease resistance ## Infecɵon Ó 2022 The Authors. Finally, it is worth mentioning that one of the major challenges plants face is defending against pathogen infection under continuous changes in nutrient availability, particularly Pi availability. Results here presented set the basis for further research to elucidate the exact mechanisms by which Pi signaling and pathogen-induced signaling interact with each other in plants. Further studies, however, should be conducted on a case-by-case basis in different plantpathogen interactions. A better understanding of these mechanisms will allow the development of novel strategies to improve disease resistance in plants. ## Experimental procedures ## Plant material and infection assays Arabidopsis thaliana (ecotype Columbia-0 [Col-0]) plants were grown in a mixture of soil:perlite:vermiculite (2:1:1) and modified Hoagland half strength medium, under neutral photoperiod (12 h light /12 h dark), at a relative humidity of 60% and a temperature of 22°C AE 2°C. The fungus P. cucumerina was grown on Potato Dextrose Agar plates with chloramphenicol (34 lg ml À1 ). Colletotrichum higginsianum was grown on Oatmeal agar plates in darkness. Fungal spores were collected by adding sterile water to the surface of the mycelium and adjusted to the desired final concentration using a B€ urker counting chamber. For infection experiments in soil-grown plants, 3-week-old plants were spray-inoculated with a spore suspension of P. cucumerina (5 9 10 5 spores/ml) or mock-inoculated. Plectosphaerella cucumerina-inoculated and mock-inoculated plants were maintained under high humidity and plant survival was assessed at 7 dpi. For infection with C. higginsianum, the fungal spores were locally inoculated (4 9 10 6 spores ml À1 ; 10 ll per leaf and five leaves per plant). The lesion area of C. higginsianum-infected leaves was measured with ImageJ software (National Institute of Health, Bethesda, MD, USA; https://imagej.nih.gov/ij/) at 7 dpi. Three independent experiments were performed with at least 12 plants per genotype in each experiment. For in vitro experiments, 2-week-old Arabidopsis plants were spray-inoculated with P. cucumerina (4 9 10 6 spores ml À1 ) or locally inoculated with C. higginsianum (5 9 10 5 spores ml À1 ). Fungal biomass was determined by quantitative PCR (qPCR) using specific primers for the corresponding fungus; the Arabidopsis UBIQUITIN21 (At5g25760) gene was used as the internal control [bib_ref] The Arabidopsis miR396 mediates pathogen-associated molecular pattern-triggered immune responses against fungal pathogens, Soto-Su Arez [/bib_ref]. PCR primers are listed in . Statistically significant differences were determined by the t-test. Elicitor treatments were performed by spraying 3-week-old plants with an elicitor extract obtained from the fungus P. cucumerina (300 lg ml À1 ) as previously described [bib_ref] Expression of the gene encoding the PR-like protein PRms in germinating maize..., Casacuberta [/bib_ref]. Three independent experiments were performed with at least 12 plants per genotype in each experiment. For Pi treatment experiments, plants were grown in vitro on meshes placed on agar plates with modified Hoagland half strength medium containing 0.25 mM KH 2 PO 4 for 1 week. Seedlings were then transferred to fresh agar medium at the desired concentration of Pi (0.05, 0.1, 0.25, or 2 mM Pi). The plants were allowed to continue growing for one more week under each Pi regime. The in vitro-grown plants were then inoculated with a spore suspension of P. cucumerina or C. higginsianum as above. The pho2 mutant, previously named ubc24 (UBIQUITIN-CONJUGATING ENZYME 24; At2g33770; Col-0 background) was obtained from the Arabidopsis Biological Resource Center (ABRC, ref. CS8508). A point mutation in the sixth exon (from G 2539 to A, relative to the translational start codon) causes an early termination at the beginning of the UBC domain, thus resulting in the loss of the ubiquitin-conjugating activity of PHO2 in the pho2 mutant . ## Plant tissue staining For trypan blue staining, leaves were fixed by vacuum infiltration for 1 h in ethanol:formaldehyde:acetic acid (80:3.5:5 v/v), stained with lactophenol blue solution for 1 h, and then washed with chloral hydrate for 15 min. Leaves were placed on glass slides with glycerol and observed using a Leica DM6 microscope under bright field conditions. For H 2 DCFDA staining, Arabidopsis leaves were placed on a solution of H 2 DCFDA (at a concentration of 10 lM), vacuum infiltrated during 5 min, and then maintained in darkness for 10 min. Two washes with distillated water were performed. Photographs were taken on a Leica DM6 microscope to visualize green fluorescence. DAB staining was also used to examine H 2 O 2 levels. For this, Arabidopsis plants were immersed in a DAB solution (1 mg ml À1 ) for 30 min with vacuum treatment, maintained during 4 h in darkness under agitation, washed with 95% ethanol for 30 min at 75°C, and observed using a Leica DM6 microscope under bright field illumination. ROS production in response to treatment with P. cucumerina elicitors (300 lg ml À1 ) was measured using the luminol method described by [bib_ref] Rapid bioassay to measure early reactive oxygen species production in Arabidopsis leave..., Smith [/bib_ref]. Measurements were carried out on a CentroXS3 LB 960 Microplate reader (Berthold Technologies, Bad Wildbad, Germany). ## Generation of transgenic arabidopsis plants For MIR399f overexpression, the miR399 precursor sequence was cloned under the control of the Cauliflower mosaic virus (CMV) 35S promoter in the PMDC32 plasmid . The plant expression vector was transferred to Agrobacterium tumefaciens strain GV301. Arabidopsis Col-0 plants were transformed using the floral dip method. Homozygosis was achieved by antibiotic selection. Segregation analysis confirmed transgene inheritance in successive generations of transgenic lines. ## Measurements of pi content and chlorophyll content The Pi content of Arabidopsis plants was determined as previously described [bib_ref] A chloroplast phosphate transporter, PHT2;1, influences allocation of phosphate within the plant..., Versaw [/bib_ref]. Chlorophylls were extracted and quantified spectrophotometrically (SpectraMax M3, Molecular Devices, San Jose, CA, USA) as previously described. ## Gene expression analyses Total RNA was extracted using TRIzol reagent (Invitrogen, Waltham, MA, USA). First-strand cDNA was synthesized from DNasetreated total RNA (1 lg) with reverse transcriptase and oligo-dT (High Capacity cDNA reverse transcription kit, Applied Biosystems, Waltham, MA, USA). RT-qPCR was performed in optical 96well plates using SYBRâ green in a LightCycler 480 (Roche, Basel, Switzerland). Primers were designed using Primer-Blast (https:// www.ncbi.nlm.nih.gov/tools/primer-blast/). The b-tubulin2 gene (At5g05620) was used to normalize the transcript levels in each sample. Primers used for RT-qPCR and stem-loop RT-qPCR are listed in . Accumulation of mature miR399f was determined by stem-loop . DNA sequencing confirmed the specificity of the PCR-amplified products. Two-way analysis of variance (ANOVA) followed by Tukey's ## Hormone determination The rosettes of 3-week-old wild-type (Col-0), miR399 OE, and pho2 plants were analyzed by LC-MS for SA, SAG, JA, and OPDA content as previously described (S anchez- [bib_ref] Mycorrhizal tomato plants fine tunes the growthdefence balance upon N depleted root..., Bel [/bib_ref]. Briefly, 30 mg of freeze-dried material was extracted with MeOH:H 2 O (30:70) containing 0.01% of HCOOH and a mixture of 10 lg L À1 of the internal standards SA-d5 and dehydro-JA (Sigma-Aldrich, St. Louis, MO, USA). Following extraction, samples were centrifuged (25 000 g, 15 min) and filtered through regenerated cellulose filters. An aliquot of 20 ll was injected into a UPLC system (Waters Acquity) interfaced with a Xevo TQ-S Mass Spectrometer (Waters, Milford, MA, USA). Hormones were quantified by contrasting with an external calibration curve of pure chemical standards of SA, SAG, JA, and OPDA. Sample separation was performed with an LC Kinetex C18 analytical column with a particle size of 5 lm, 2.1 9 100 mm (Phenomenex, Torrance, CA, USA). Chromatographic and MS conditions were as described in S anchez- [bib_ref] Mycorrhizal tomato plants fine tunes the growthdefence balance upon N depleted root..., Bel [/bib_ref]. At least six biological replicates were analyzed per genotype and condition, each replicate consisting of leaves from at least three independent plants. The plant material was lyophilized prior to analysis. Two-way ANOVA followed by Tukey's HSD test was used to analyze data. ## Accession numbers b-Tubulin 2 (At5g62690); PHO2 (At2g33770); PR1 (At2g14610); NPR1 (At1g64280); PAD4 (At3g52430); ERF1 (At3g23240); PDF1.2 (At2g26020); MYC2 (At1g32640); VSP2 (At5g24770); RBOHD (At5g47910); Ubiquitin 21 (At5g25760). # Acknowledgments We thank A. Molina and R.J. O'Connell for providing the P. cucumerina and C. higginsianum strains and Gl oria Escol a for assistance in parts of this work. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI). This research was supported by the Ministry of Science, Innovation and Universities/FEDER/Innovation-State Research Agency (AEI) (grant RTI2018-101275-B-I00) to BSS, the Research Promotion Plan from the Universitat Jaume I (UJI-B2019-2), and the Scientific Instrumentation Central Service from the Universitat Jaume I to VF. We acknowledge financial support from the Spanish Ministry of Science/AEI through the "Severo Ochoa Programme for Centres of Excellence in R&D" SEV-2015-0533 and CEX2019-000902-S, and the CERCA Programme/"Generalitat de Catalunya". BVT was a recipient of a fellowship from the Ministry of Economy, Industry and Competitiveness/AEI (BES-2016-076289). MBP was a recipient of a scholarship from La Caixa Foundation. # Author contributions BSS and TJC devised the research project. MB, BVT, and BSS designed the experiments and analyzed the data. BVT conducted most experiments and prepared the figures. MBP performed infection experiments with P. cucumerina. HMC collaborated in expression analyses and ROS detection. VF performed hormone analyses. BSS and BVT wrote the article with further contributions from MB. All authors supervised and complemented the writing. BSS agrees to be responsible for contact and ensures communication. ## Conflict of interest The authors declare that they do not have competing interests. # Data availability statement All relevant data can be found within the published article and its supporting material. ## Supporting information Additional Supporting Information may be found in the online version of this article. [fig_ref] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P [/fig_ref]. Phenotypical analysis of miR399 OE plants. [fig_ref] Figure 2: Resistance of miR399 OE plants to infection by the hemibiotrophic fungus C [/fig_ref]. Analysis of pho2 mutant plants under non-infection conditions. [fig_ref] Figure 3: Resistance to infection by fungal pathogens in pho2 plants [/fig_ref]. ROS accumulation in leaves of high Pi plants (miR399 OE, pho2 plants) under non-infection conditions and upon infection with P. cucumerina as determined by DAB staining.. ROS accumulation in leaves of high Pi plants (miR399 OE, pho2 plants) in response to treatment with P. cucumerina elicitors as determined by H 2 DCFDA staining. . ROS production in leaves of miR399 OE, pho2, and wild-type plants in response to treatment with P. cucumerina elicitors as determined using the luminol assay. ROS production in wild-type plants that have been grown under different Pi supply was also determined using the luminol assay. . Expression of defense-related genes in wild-type Arabidopsis plants that have been grown under different Pi supply (0.05, 0.1, or 0.25 mM). . List of oligonucleotides. [fig] Ó 2022: The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452-469 type plants, as revealed by quantification of diseased leaf area and fungal biomass (Figure 2a). As observed in infection assays with P. cucumerina, the C. higginsianum-infected miR399 OE plants exhibited groups of dead cells in their leaves (Figure 2b). [/fig] [fig] Figure 1: Resistance of miR399 OE plants to infection by the necrotrophic fungus P. cucumerina. [/fig] [fig] Figure 2: Resistance of miR399 OE plants to infection by the hemibiotrophic fungus C. higginsianum. [/fig] [fig] Figure 3: Resistance to infection by fungal pathogens in pho2 plants. Three-week-old mutant plants were inoculated with fungal P. cucumerina spores. [/fig] [fig] Figure 4: Acumulation of precursor (pre-miR399), mature (miR399), and PHO2 transcripts in wild-type plants in response to inoculation with P. cucumerina spores or treatment with P. cucumerina elicitors. The accumulation of pre-miR399f and mature miR399f sequences was determined by RT-qPCR and stem-loop RT-qPCR, respectively, at the indicated times after inoculation with fungal spores (a) or elicitor treatment (b). At 48 and 72 hpi with P. cucumerina spores, pre-miR399 levels increased 4.5-and 6.0-fold, respectively. At the same time points, miR399f levels increased 2.0-and 9.0-fold. Elicitor treatment elevated the level of pre-miR399f accumulation 5.0-and 267-fold at 90 and 120 min of treatment, respectively. MiR399f levels increased 20-and 147-fold in response to elicitor treatment (90 and 120 min, respectively). Four biological replicates and three technical replicates per time point were assayed. Statistically significant differences were determined by ANOVA followed by Tukey's HSD test, where different letters represent statistically significant differences. [/fig] [fig] Figure 6, Figure 7: Expression of defense genes in miR399 OE and pho2 plants. Transcript levels were determined by RT-qPCR in mock-inoculated or P. cucumerina-inoculated plants at 24 h post-inoculation (black and gray bars, respectively). The expression values were normalized to the Arabidopsis b-tubulin2 gene (At5g62690). (a) Expression of genes involved in the SA pathway (PR1, NPR1, PAD4). (b) Expression of genes in the ERF branch of the JA pathway (ERF1, PDF1.2). (c) Expression of genes in the MYC branch of the JA pathway (MYC2, VSP2). Three independent experiments (with 12 plants per genotype) were examined, with similar results. Bars represent mean AE SEM. Different letters represent statistically significant differences (ANOVA followed by Tukey's HSD test; P < 0.05). Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452-469 pathogens with necrotrophic (P. cucumerina) and hemibiotrophic (C. higginsianum) lifestyles. On the other hand, resistance in Arabidopsis plants overaccumulating Pi correlated well with upregulation of defense-related genes under normal growth conditions (i.e., in the absence of pathogen infection). Additionally, we show that P. cucumerina infection is accompanied by an increase in miR399 accumulation. Not only pathogen infection, but also treatment with fungal elicitors results in higher levels of miR399. The observed elicitor-responsiveness of MIR399 Levels of SA, SAG, JA, and OPDA in leaves of Arabidopsis plants that overaccumulate Pi (miR399 OE, pho2, and wild-type plants). (a) Hormone levels were measured in mock-inoculated and P. cucumerina-inoculated miR399 and pho2 plants at 48 hpi. (b) Hormone levels in wild-type plants that have been grown at the indicated Pi supply conditions (0.05, 0.1, and 0.25 mM Pi). Plants were grown as indicated in Figure 5. Three independent experiments (with 12 plants per genotype) were examined, with similar results. Bars represent mean AE SEM. Different letters represent statistically significant differences (ANOVA followed by Tukey's HSD test; P < 0.05). Ó 2022 The Authors. The Plant Journal published by Society for Experimental Biology and John Wiley & Sons Ltd., The Plant Journal, (2022), 110, 452-469 [/fig] [fig] Figure 8: Proposed model to explain how Pi content and defense responses are integrated for modulation of resistance to infection by fungal pathogens in Arabidopsis. Treatment with high Pi, MIR399 overexpression, and loss-offunction of PHO2 would trigger Pi accumulation, which, in turn, would increase ROS and hormone (SA and JA) levels in Arabidopsis leaves for the induction of genes involved in the SA and JA signaling pathways. Upon pathogen infection, the expression of genes involved in the SA pathway and the ERF1/PDF1.2 branch of the JA pathway would be further induced, while genes in the MYC2/VSP2 branch of the JA pathway are repressed. [/fig]

Strategies for in vitro engineering of the translation machinery Engineering the process of molecular translation, or protein biosynthesis, has emerged as a major opportunity in synthetic and chemical biology to generate novel biological insights and enable new applications (e.g. designer protein therapeutics). Here, we review methods for engineering the process of translation in vitro. We discuss the advantages and drawbacks of the two major strategies--purified and extract-based systems--and how they may be used to manipulate and study translation. Techniques to engineer each component of the translation machinery are covered in turn, including transfer RNAs, translation factors, and the ribosome. Finally, future directions and enabling technological advances for the field are discussed. # Introduction The translation machinery--the ribosome and associated factors necessary for protein biosynthesis--polymerizes L-␣-amino acid building blocks into proteins according to instructions presented in messenger RNA (mRNA) and defined by the genetic code . Given the redundancy of the genetic code (i.e. 64 codons encode only 20 amino acids), there has long been interest in understanding how one might re-engineer the genetic code to incorporate monomers with novel side chain chemistries or backbones. Key among efforts to reprogram the genetic code are pioneering studies that have shown the flexibility of the translation system to incorporate non-canonical amino acids (ncAA) into biopolymers using genetic code expansion approaches (6-10) . Recently, substantial advances have been made in the considerably more challenging task of incorporating monomers with novel backbones, including N-methyl-, ␣-hydroxy acid, D-␣-and ␤-amino acids, as well as polypeptoidsand even foldamers. Such monomers allow modulation of not just side chain chemistry but polymer folding and stability properties as well . Considering these possibilities, expanding the genetic code for the synthesis of novel sequence-defined polymers has emerged as a major opportunity in synthetic and chemical biology. Engineering the translation machinery is a complex and formidable challenge for many reasons. Here, we highlight three. First, translation involves the interplay of dozens of individual proteins and RNAs, and due to the challenge of optimizing all these components simultaneously, most studies have focused on altering only one or two components at a time. Second, protein translation with non-canonical monomers often suffers from poor efficiencies and low yields of full-length product, especially when incorporating multiple, distinct ncAAs. Third, biological constraints limit the scope of permissible engineering possibilities to expand ncAA diversity. An especially challenging constraint is the limited mutability of the ribosome, since ribosome function must be preserved to maintain cell viability. This restricts the mutations that can be made to the ribosome, thus excluding many ribosomal RNA (rRNA) genotypes that may enable new ribosome function, but are incompatible with the viability of living cells. To address the complex issues above, new tools are needed to derive general paradigms for engineering translation systems. Though most efforts to engineer the translation system have been pursued in vivo (i.e. in living cells), in vitro (i.e. cell-free) approaches have several key benefits. First, they do not suffer from cell-viability constraints, facilitating the use of toxic genotypes (e.g. orthogonal translation system components required to expand the genetic code and incorporate ncAAs) and non-physiological reaction conditions. Second, cell-free systems allow precise control of reaction conditions and permit the addition A B C . Conceptual goals for engineering templated polymer production by the ribosome. (A) The standard genetic code enables the polymerization of canonical amino acids with a diversity of 20 proteinogenic side chains (blue shaded circles). Despite enabling the evolution of life and having been harnessed for societal needs (e.g. recombinant protein production of protein therapeutics like insulin), protein biosynthesis in nature uses limited sets of protein monomers, which results in limited sets of biopolymers (i.e. proteins). (B) First-generation genetic code expansion facilitates the incorporation of L-␣-amino acids with a vast array of chemical side chains into proteins (pink circles). Site-specific incorporation of up toinstances of a single ncAA in a single polypeptide chain has been reported. (C) Next-generation genetic code expansion involves the incorporation of monomers with both non-canonical side chains and backbones (multi-colored stars). Engineering of all aspects of the translation apparatus will be required to generate systems capable of efficiently carrying out polymerization of these exotic new molecules. and removal of individual components to study their effects on translation. Third, they enable rapid, automationassisted assembly of reactions from individual components for efficient system optimization. Taken together, these features provide a freedom of design and control that make cell-free systems an attractive complement to cellular approaches for studying and engineering translation. This review aims to provide an overview of recent advances for engineering the translation machinery in vitro. We begin by covering the two general platforms for in vitro protein translation: the PURE system (i.e. protein synthesis using purified recombinant elements) and extract-based systems. We then examine strategies for engineering each non-ribosomal component of the translational system, including transfer RNAs (tRNAs) and translation factors. We next cover strategies for the reconstitution and in vitro synthesis of the ribosome, which set the stage for engineering the central catalyst of translation. Finally, we review recent technological advances that will impact in vitro translation engineering and discuss the future outlook of the field. Overall, this review is intended to provide a focused per-spective on the past, current, and future challenges of in vitro translation engineering for those researchers wishing to learn about and influence this rapidly developing field. ## In vitro protein translation platforms In vitro translation systems facilitate the biosynthesis of recombinant proteins without using intact cells. In recent years, improvements in such systems have enabled accurate and efficient incorporation of ncAAs into proteins for genetic code expansion. Two main platforms have been developed: the PURE system and the extract-based system. ## The pure translation system In the PURE system, all the translation factors, tRNAs, components for mRNA template generation, and ribosomes are individually purified from cells and assembled in vitro to create a translationally competent environment, left). This strategy enables the user to define the concentrations and genotypes of all components in the translation reaction. The exquisite control afforded by the PURE system has spawned a variety of synthetic biology platforms which leverage this capability. For example, Suga et al.'s pioneering efforts have used the flexibility for genetic code reprogramming available in the PURE system for highly efficient sense and non-sense suppression in incorporating ncAAs into peptidomimetic drugs. Additionally, Forster et al. showed the ability to program peptidomimetics by translating genetic codes designed de novo. However, this approach entails several challenges that have been addressed to varying degrees. First, determining the ideal concentration of each translation component is a difficult optimization problem. A systematic analysis of interactions between the concentrations of 69 translation components enabled optimization of the concentration of those components in the PURE system. Subsequent improvements resulted in protein yields of 4.4 g/l of ␤-galactosidase in a semi-continuous reactionor a 5-fold improvement in luciferase production from translation factor optimization and a further ∼2-fold improvement by replenishing six small molecule substrates. A second challenge is the high relative cost of the PURE system compared with extract-based systems. Estimates suggest that PURE is ∼2 orders of magnitude more expensive per gram of protein produced than extract-based approaches. Recently published techniques to simplify the process of generating PURE system components can substantially reduce costs and labor, but since all these techniques use one-pot purifications, they also necessarily entail some loss in control and modularity over translation components. ## Extract-based systems The history of extract-based in vitro translation systems is rooted in the origins of molecular biology, as such systems were used to elucidate the genetic code. Recently, extract-based protein synthesis methods have en- In vitro protein synthesis systems facilitate translation system engineering. Two strategies exist for enabling protein translation in vitro: the PURE system and extract-based systems. In the PURE system (left), each unique component of the translation apparatus is individually purified from cells, including the aminoacyl-tRNA synthetases, tRNAs, translation factors, and ribosomes. In order to reconstitute a functional translation system, these components are then recombined together with amino acids, energy substrates, cofactors, salts, a template for a protein of interest (POI), and T7 RNA polymerase (RNAP) to generate mRNA template. Importantly, this methodology enables precise optimization of component concentrations and the ability to leave out certain components and replace them with modified components to modulate translation apparatus function. In contrast, extract-based systems (right) entail a simpler protocol for the preparation of a crude cellular extract containing all the necessary components. joyed a resurgence in interest driven by advances in system capabilities such as high-level protein expression (> g/l) for prototyping and characterizing biological systems, on-demand biomanufacturing, glycoprotein synthesis, molecular diagnostics (60-64) and education, among others (reviewed in. While a variety of cell-free reaction preparation methods exist, each generally involves lysis and the extraction of the crude intracellular milieu, supplementation with enhancing components such as cofactors and an energy source, and protein synthesis from a DNA template, right). As a platform for engineering translation, the primary advantage of extract-based methodologies is the ability to obtain the entire complement of translation machinery components with a simple extraction to remove cell wall debris and chromosomal DNA. This method also retains ancil-lary components that aid functional protein synthesis, such as recycling enzymes, metabolic enzymes, chaperones, and foldases. These components may account for the ability of extract-based systems to produce more protein per ribosome than the PURE approach. While crude extract-based systems offer simplicity of preparation, the difficulty of completely defining the translational environment is a drawback. Exerting greater control over extract-based systems entails more involved extract processing, including selective depletion of components of the translation machinery. For example, depletion of tRNAs via degradationor DNA-hybridization chromatography, or inactivation of tRNAs via sequestration using synthetic oligonucleotidescan be used to reassign the meaning of sense codons in extracts. Similarly, removal of native ribosomes via ultracentrifugation (i.e. 150 Nucleic Acids 000 × g) is the basis for a platform that can build ribosomes in vitro. Finally, while this strategy has not been implemented in bacterial extract to our knowledge, translation factors may be depleted to create a platform to study and engineer their function. Strain engineering to improve extract-based systems. Strain engineering is critical to create extracts which are optimized for high-level in vitro protein production. Genomic recoding, in which codons are systematically removed from the genome, is especially useful in engineering alterations to the genetic code in extract-based systems. The systematic global recoding of a codon to a synonymous alternative is required before its meaning can be changed without incurring detrimental or lethal effects. The power of recoding for in vitro ncAA incorporation was first demonstrated with the incorporation of the p-acetyl-Lphenylalanine (pAcF) at up to five sites in superfolder green fluorescent protein (sfGFP) in a partially recoded, release factor 1 (RF1) deficient strain, in which 13 occurrences of the amber stop codon (UAG) were reassigned to the synonymous UAA codon. Later, a fully recoded strain lacking all amber codons (C321. A) with knockouts of RF1 and the phosphoserine (Sep) phosphatase SerB and introduction of a Sep orthogonal translation system (OTS) enabled site-specific incorporation of multiple Sep residues in a single protein in extract. This provided new insights into the role of serine phosphorylation on MEK1 kinase activity and increased the resolution at which phosphorylation-induced effects on protein structure and function can be defined, manipulated, and understood. Optimization of the fully recoded C321. A -including the knockout of the genes endA, gor, rne and mazFimproved cell-free protein synthesis yields to >1.7 g/l in batch reactions and facilitated the incorporation of 40 identical pAcF residues site specifically into an elastin-like polypeptide with high ( ≥98%) accuracy of incorporation. More recently, Des Soye et al. modified the aforementioned optimized, fully recoded strain of Escherichia coli to express T7 RNA polymerase and enable high-yielding (∼2.7 g/l) cell-free transcription and translation reactions without exogenous polymerase addition. These yields outperform the best reported expression of proteins with single or multiple ncAAs in vivo. Overall, the ease of use and lower cost of extract-based protein synthesis makes it an attractive platform, and methods to gain greater control over reaction conditions are expanding the range of useful applications. Taken together, the intensive development of both PURE and crude extract-based in vitro translation systems mean that an appropriate cell-free system is available for most engineering projects leveraging bacterial translation (nonbacterial cell-free translation systems are not covered in this review). However, when the goal is expansion or modification of the genetic code, the individual components of the translation machinery require special consideration. We next discuss the non-ribosomal components of the translation apparatus, what is known about their function, and how they may be engineered to enable in vitro alteration of the genetic code. ## Trna engineering Aminoacyl-tRNAs (aa-tRNAs) are at center stage during protein translation. They function as adapter molecules, enforcing the genetic code by recognizing the sequence information of an mRNA template and delivering their charged amino acid for incorporation into the growing polypeptide chain. More specifically, each tRNA must be (i) selectively charged by its cognate aminoacyl-tRNA synthetase (AARS), (ii) efficiently bound in its aminoacylated form by EF-Tu for transport to the ribosome, and each must function optimally in translation by (iii) binding to the ribosomal A site, (iv) enabling peptidyl transfer, (v) translocating to the ribosomal P site, (vi) facilitating another acyl transfer reaction, and (vii) finally releasing from the ribosome. Below, we describe how tRNAs can be made, charged for use in in vitro reactions, and tuned for enhanced translation activity. ## In vitro transcription of trnas Synthesis of tRNAs for cell-free protein synthesis can be done using in vitro transcription (IVT), wherein unmodified tRNA is synthesized by T3, T7 or SP6 bacteriophage systems. The T7 RNA polymerase (T7 RNAP) is most commonly used. T7 RNAP accepts linearized plasmid DNA, PCR products, or synthetic oligonucleotides templates containing a T7 promoter, and synthesis kits are commercially available. Importantly, the +1 nucleotide of the T7 promoter is usually a guanine or adenine and will be the template for the first nucleotide of the transcribed RNA. Moreover, two guanines following the +1 nucleotide greatly improve transcription yields. Due to these constraints, not every tRNA can be easily synthesized using T7 RNAP, and RNA yields vary with the sequence of the tRNA. To overcome this problem, self-excising ribozymes can be inserted between T7 RNAP promoter and tRNA template, enabling efficient transcription and control of the exact 5 sequence. A similar trick can be applied to prevent T7 RNAP-mediated overextension at the 3 -end which would otherwise occlude the terminal adenine required for aminoacylation. Alternatively, DNA templates modified with methoxy moieties at the ribose C2 position of the last two nucleotides help prevent non-templated nucleotide addition at the 3 end. Although these tRNAs lack post-transcriptional modifications (88), a recent tour de force demonstrated that most of the 48 E. coli tRNAs can be synthesized using T7 RNAP and are functional in translation. Only five tRNAs (the isoacceptors for Glu, Asn and Ile) appear to require post-transcriptional modifications for activity. ## In vitro trna aminoacylation methods To introduce ncAAs site-specifically into polypeptides, tRNAs need to be 'misacylated' with monomers beyond their native amino acids, which enables reassignment of codons to chemical substrates of interest. Four methods are used to generate 'misacylated' tRNAs in vitro: enzymatic aminoacylation via engineered AARSs, chemical-enzymatic aminoacylation, chemical modification of aminoacylated cognate amino acids, and flexizymecatalyzed aminoacylation. tRNA aminoacylation via engineered orthogonal AARS/tRNA pairs. In their native context, tRNAs are enzymatically aminoacylated with their cognate amino acids by highly specific AARS enzymes. These enzymes recognize their cognate tRNA substrates via identity elements in the tRNA's acceptor stem, D-loop, variable loop, and the anticodon loop. The amino acid specificity is determined by the amino acid binding pocket and, in case of some AARSs, an additional editing domain which hydrolyzes misacylated tRNAs to ensure an accuracy in aminoacylation of at least 10 000:1. Because of this stringent quality control, many ncAAs are not readily accepted by AARSs. To overcome this limitation, directed evolution has been used to generate orthogonal (o)-AARS/o-tRNA which charge the o-tRNA with desired ncAAs. In bacteria, the most widely used pairs are derived from the tyrosyl-(Tyr)RS/tRNA Tyr pair from Methanocaldococcus jannaschii or the native amber suppressor pyrrolysyl-(Pyl)RS/tRNA Pyl pair from Methanosarcina barkeri. Original innovations pioneered by Schultz and colleagueshave been adapted for many ncAAs, leading to, for example, M. jannaschii TyrRS/tRNA Tyr pairs capable of installing diverse tyrosine derivatives (2), PylRS/tRNA Pyl pairs incorporating lysine, phenylalanine and pyrrolysine derivatives, as well as click chemistry-reactive ncAAs (91), S. cerevisiae TrpRS/tRNA Trp incorporating tryptophan derivatives, and P. horikoshii ProRS/tRNA Pro allowing incorporation of proline derivatives. More recent work guided by crystal structures, genome engineering methods, and next generation sequencing, have pushed the limits of generating highly selective and orthogonal AARS/tRNA pairs that enhance the insertion of ncAAs into proteins. These include: compartmentalized partnered replication (CPR) (93), phage-assisted continuous evolution (PACE) (95), parallel positive selections, and multiplex automated genome engineering (MAGE) (97,98), among others. While many o-AARS/o-tRNAs pairs have been evolved in vivo, all of them can be used in vitro by adding them in purified form to either extract-based systems or the PURE system. In the case of extract-based systems, they also can be expressed in the extract source strain, circumventing time-consuming purification steps (99). Despite many successful examples of changing the amino acid specificity of AARSs, these engineered enzymes still generally suffer from lower catalytic activity relative to their native counterparts. However, the use of these components in vitro provides a means to overcome catalytic inefficiencies, as the concentration of the o-AARS, the o-tRNA, and the ncAA can all be increased as required to attain robust ncAA incorporation. Accordingly, several groups have used extract-based systems with o-AARS/o-tRNA pairs to produce proteins containing ncAA via site-specific incorporation. In one example, Albayrak and Swartz demonstrated the potential of crude extracts to produce ncAA-containing proteins in higher yields by co-expressing an o-tRNA for amber stop codon suppression together with the target protein from one DNA template. In this way tRNA limitations could be overcome. While such innovations offer a work-around for low enzyme efficiencies, engineering orthogonal translation systems with high activity and specificity for a unique ncAA remains a significant systems-level challenge. Chemical-enzymatic aminoacylation of tRNA. While AARS engineering is one approach to preparation of 'misacylated' tRNAs, this task can alternatively be accomplished using one of two chemical methods. The first approach combines chemical aminoacylation and enzymatic oligonucleotide ligation. This method involves synthesis and chemical aminoacylation of the hybrid dinucleotide 5 -phospho-2 -deoxyribocytidylylriboadenosine (pdCpA) using an activated amino acid donor with an N-protected group followed by HPLC purification, concomitant ligation of the aminoacylated pdCpA to a truncated tRNA lacking the 3 terminal CA via T4 RNA ligase, and deprotection to liberate the free ␣-amino group (107-110), left). This approach is advantageous because it allows one to work with many different tRNAs to determine how the identity of the tRNA impacts the efficiency of in vitro translation. Additionally, a recent report suggests the potential for improved translation activity with artificial aminoacyl-tRNA substrates made with an N-nitroveratrylooxycarbonyl (N-NVOC)-monomer-pCpA synthesis method. In the second method, canonical amino acids loaded onto tRNAs by their cognate AARS are subsequently chemically modified into ncAAs. For example, Fahnestock and Rich generated phenyllactyl-tRNAs through deamination of Phe-tRNAs with nitrous acid. Similarly, Merryman and Green generated tRNAs bearing N-monomethyl amino in a three-step process: protection of the ␣-amino group of the aa-tRNA using o-nitrobenzaldehyde, reductive methylation using formaldehyde, and deprotection by UV radiation to liberate the free ␣-N-methyl-amino group, center). Although these methods made ncAAs with bulky side chains accessible (such as glycosylated derivatives and larger organic fluorescent dyes, and are applicable to nearly every ncAA in principle, they are technically demanding, laborious and often yield poor incorporation results due to the generation of a cyclic tRNA by-product which inhibits ribosomal peptide synthesis. Flexizyme-catalyzed aminoacylation. Beyond proteincatalyzed and chemical charging approaches, ribozymecatalyzed approaches also exist for acylating ncAAs to tRNAs. Specifically, small artificial ribozymes (44-46 nt) called Flexizymes (Fx) can be used to generate ncAA-tRNAs. Flexizymes originate from an acyl-transferase ribozyme (ATRib) capable of transferring N-biotin-Phe from the 3 -end of a short RNA to its own 5 -OH group. Through directed evolution and sequence optimization, ATRib was evolved into a family of three different Fxs (eFx, dFx and aFx) with different affinities to specific substrate-activating groups (120): eFx is used to acylate tRNAs with cyanomethyl ester (CME)-activated acids containing aryl functionality, dFx recognizes dinitrobenzyl ester (DNBE)-activated non-aryl acids, and aFx recognizes the hydrophilic activating group (2-aminoethyl)amidocarboxybenzyl thioester (ABT) which allows it to charge compounds with poor solubility in water. Flexizymes selectively aminoacylate the 3 -OH of any tRNA, regardless of the body and anticodon sequences, with a broad range of carboxylic acids, including D-, ␤-, ␥ -(122) and other non-canonical amino acids (123) as well as N-alkylated amino acidsand even hydroxy acids, benzoic acids (125), exotic peptides (126) and foldamers. With the Flexizyme approach, a great variety of amino acids can be assigned to any tRNA as long as the amino acid side chain is stable during the esterification reaction and the monomer can be attached to the activated leaving group. Hence, in principle, the combination of Fx-catalyzed tRNA charging with an appropriate in vitro translation system allows near-total freedom in reassigning any codon with any ncAA. The most commonly used custom-made reconstituted translation system of this kind is called FIT (Flexible In-vitro Translation) system. ## Trna engineering for improved ncaa incorporation The tuning of the translation machinery that has occurred through evolution has yielded tRNAs and trans-lation factors--in particular Elongation Factor-Tu (EF-Tu)--with thermodynamic compensation interactions that are tuned to match canonical amino acids with cognate tR-NAs. As a result, engineering aminoacyl-tRNAs with optimal loading properties for EF-Tu is important. To this end, key targets for tRNA engineering include mutations at or near the anticodon recognition domain of the AARS, as well as the T-stem region of the tRNA, which interacts with residues from the ␤-barrel domain 2 and the GTPase domain of EF-Tu (98). In one example, Guo et al. evolved M. jannaschii tRNA Tyr CUA variants for amber suppression by targeting regions implicated in EF-Tu binding. Modifications in EF-Tu binding regions of tRNA Pyl also improved ncAA incorporation efficiencies using an o-PylRS/ o-tRNA Pyl pair. The best variant of this study facilitated a 3-fold improvement in suppressing one amber codon and a 5-fold improvement when suppressing two. Complementary approaches involving engineering of EF-Tu itself have also been explored (discussed below). ## Translation factor engineering While the tRNAs are the adapters essential for decoding the mRNA message, various protein factors orchestrate the process of translation. They are responsible for initiating translation (IF1, IF2, IF3), choreographing transla- tion through normal (EF-Tu, EF-G) or challenging (EF-P) sequences, and terminating translation at the three stop codons (RF1, RF2). Considering the integral roles these factors play in the process, it is unsurprising that they are attractive targets for engineering translation in vitro. The following section surveys the roles of translation factors in vitro, summarizes work that has helped elucidate their functions, and describes their roles in promoting optimal translation in cell-free systems. ## Translation initiation engineering In bacterial systems, translation is canonically initiated by the initiator tRNA (tRNA fMet CAU ) which has been first charged with the initiator amino acid methionine (Met) and then formylated at the ␣-amino group on Met by methionyl-tRNA synthetase (MTF) to form fMet-tRNA fMet CAU . The fMet-tRNA fMet CAU is then recruited by initiation factor 2 (IF2) to the 30S ribosomal subunit in the presence of all three initiation factors to form the 30S initiation complex. Considering this complex assembly of specialized initiator molecules, one might expect that engineering trans-lation initiation would be a daunting task. However, pioneering works from the Schulman group demonstrated that protein synthesis can be initiated with non-methionine amino acids charged to tRNA fMet with alternative anticodons. This implies that aspects of the tRNA fMet (and not the attached amino acid or anticodon loop) are the primary selection determinant of IF2 for translation initiation. Later work using the PURE system demonstrated that 11 of the 19 amino acids other than Met were capable of initiating translation with greater than 50% the efficiency of wildtype initiation when charged to tRNA fMet CAU. A number of functionalized N ␣ -acyl groups were also accepted, enabling spontaneous cyclization when paired with a C-terminal cysteine. D-amino acids acylated to tRNA fMet CUA have also been demonstrated to competently initiate translation --especially when pre-acylated to mimic the formylated state--demonstrating that the chirality of the amino acid is not a requirement for translation initiation. The finding that formylation (or its mimic, acylation) improves but is not strictly required for translation initiation supports the hypothesis that the primary function of formylation is to discriminate against tRNA fMet CAU binding to EF-Tu. This Nucleic Acids Research, 2020, Vol. secures the role of tRNA fMet CAU as solely an initiator tRNA by preventing sequestration by the highly abundant EF-Tu. Beyond compatibility with non-canonical monomers, even short peptides acylated to tRNA fMet CAU are capable of initiating translation -in some cases reported with even greater than wild-type efficiency. This technology was extended to enable the N-terminal incorporation of short peptide foldamers which were then cyclized using established techniques to generate peptides with defined and diverse structures. Notably, altering IF2 concentration did not improve foldamer incorporation in this study, despite the direct interaction between IF2 and tRNA fMet CAU in the initiation process, suggesting that IF2 concentration is not limiting in 30S initiation complex formation. Supplementation of other initiation factors (IF1, IF3) or engineering of the ribosomal RNA itself may provide interesting targets for improving initiation with noncanonical substrates. ## Translation elongation engineering After translation initiation, all the remaining amino acids in the production of a given polypeptide are added in the elongation phase. Thus, while translation initiation is critical, in vitro translation engineering must necessarily also focus on improving and modifying elongation, as this process is essential for the diversity of available monomers and the efficiency of polymer production. Such engineering efforts in elongation have significant barriers to overcome since, in contrast to initiation, elongation discriminates against some non-canonical monomers (e.g. D-amino acids). With 10 or more copies present per ribosome, elongation factor EF-Tu is the most abundant protein in E. coli and is especially critical for efficient translation. EF-Tu is responsible for shuttling aminoacylated tRNAs to the ribosome while protecting against premature cleavage of the amino acid. The energetic interactions of each tRNA with EF-Tu combine with those of its cognate aminoacylated amino acid to produce a similar binding energy between each correctly aminoacylated tRNA and EF-Tu. This 'goldilocks' energy is strong enough to promote binding and protection of the aa-tRNA by EF-Tu, but weak enough to enable efficient release of the aa-tRNA for decoding in the A-site of the ribosome during elongation. The importance of this interaction for translation elongation has made EF-Tu an attractive target for modification to engineer non-canonical monomer incorporation -especially since monomers with large or negatively charged side chains are known to reduce binding affinity to EF-Tu. Foundational work demonstrated that the incorporation efficiency of ncAAs with bulky side chains and the preparation of tRNAs charged with ncAAs could be improved by utilizing an engineered version of EF-Tu with an enlarged binding pocket in the PURE system. In a series of similar efforts, randomization of the amino acid binding pocket of EF-Tu permitted the incorporation of the negatively-charged phosphoserine in vivo, later enabling milligram quantity production of phosphoproteins in vitro using cell extract from the recoded strain C321. A, and increasing incorporation of p-azido-phenylalanine into proteins. A similar strategy was used to improve the EF-Tu guided incorporation of selenocysteine in the SECIS-free selenoprotein synthesis system. Later, the tRNA in this system was modified to encourage productive binding to EF-Tu and efficient decoding, as in the case of the engineered tRNAUTuX which was developed to improve selenocysteine incorporation and reduce serine misincorporation in vitro. Despite these key successes, engineering the binding pocket of EF-Tu is not always an effective strategy, and was detrimental to the incorporation of D-amino acids in one study. Future projects in this space may benefit from the throughput and ability to test many combinations of variants provided by in vitro systems. During the elongation process, peptide bond formation efficiencies depend on the steric and reactive properties of the AA-tRNAs in the ribosomal active site. Hence, incorporation of several ncAAs with non-canonical backbones such as D-␣-and ␤-amino acids have suffered from low efficiencies. EF-P is a bacterial translation factor that accelerates peptide bond formation between consecutive prolines. It has been shown that the requirement of EF-P stems from the imino acid's low reactivity, steric orientation, and rigidity in the ribosomal active site, and not from a requirement of tRNA Pro. Furthermore, it has also been shown that the identity element for EF-P binding is the 9-nt D-loop found in tRNA Pro isoacceptors, and not proline itself. EF-P then binds to the P-site peptidyl-Pro-tRNA Pro to promote peptide bond formation with the A-site Pro-tRNA Pro , preventing ribosome stalling and accompanied peptidyl-tRNA drop-off. Given the nonspecificity for the charged amino acid and its ability to facilitate translation of challenging sequences, the potential of EF-P to facilitate translation engineering is compelling. The complex interplay between the elongation factors, tRNAs, and the ribosome highlight the need for multicomponent engineering of the translation system. The flexibility of cell-free systems may offer some advantages here. For example, Katoh et al. generated a hybrid tRNA, called tRNA Pro1E2 , consisting of the T-stem motif of E. coli tRNA Glu as well as the D-arm motif of E. coli tRNA Pro1 to facilitate improved synthesis efficiencies based on tighter binding to EF-Tu. The T-stem motif derived from tRNA Glu has a high binding affinity to EF-Tu, so by combining this motif with the D-arm motif of tRNA Pro1 , the authors compensated for the general low affinity of D-␣and ␤-aminoacyl-tRNAs toward EF-Tu, improving peptide bond formation. This enabled enhanced incorporation of D-amino acids and consecutive incorporation of ␤-amino acids, especially when adding EF-P to the translation system. Combining these efforts with engineering of EF-Tu may further improve this system. ## Translation termination engineering Translation is terminated at the codons UAA, UAG and UGA by the release factors RF1 (UAA, UAG) and RF2 (UAA, UGA). Since genetic code expansion efforts have traditionally targeted the rare UAG stop codon for recoding, the deletion of RF1 was an important goal to minimize errant truncation at UAG codons intended for recoding. In one approach, ribosome libraries are built in vivo and entail transformation of a library of rRNA variants, expression of those variants in living cells, and purification of fully assembled ribosomes for in vitro manipulation (top). One drawback of this method is that dominant lethal genotypes--those which kill any cell in which they are expressed--will not be present in the final library (grayed out cells). In contrast, methods for building ribosomes purely in vitro can avoid this constraint, enabling the construction of many ribosomal variants which may be lethal in vivo (purple). It is possible, however, that this library may be missing ribosomal variants which have difficulty assembling properly in vitro (yellow). This goal was first achieved by partial removal, and later, as described above, complete removal (76) of the UAG codon from the E. coli genome, which permitted the genomic deletion of RF1, enabling complete reassignment of the amber codon translation function in strain C321.ΔA. While genome-wide substitution of stop codons by defined synonyms recoding has made a tremendous impact on efforts to site-specifically incorporate ncAAs into proteins, engineering of release factors themselves has so far been limited. Toward the goal of making an 'omnipotent' release factor which terminates at all three stop codons, modification of RF2 at position 213 with the corresponding RF1 residue reduced its discrimination against termination at the UAG stop codon, though with greatly reduced overall termination efficiency in in vitro competitive peptide release experiments. In the opposing direction, one could imagine reducing the number of viable stop codons targeted by an orthogonal release factor to open multiple stop codons for recoding in vitro, or even engineering the RF to target a sense codon. ## Engineering the ribosome As the central catalytic machine facilitating peptide bond formation, the ribosome is an obvious target for engineering translation. Though it has evolved to build polypeptides out of the canonical set of 20 amino acids, the wildtype ribosome is also capable of producing polymers with non-peptide backbones (e.g. polyesters), and has even incorporated select exotic monomers and foldamers. Although many types of monomers can be incorporated into a growing polymer chain by the ribosome, incorporation of backbone-modified monomers (e.g. cyclic, ␤or D-amino acids) remains limited in the total peptide length and incorporation efficiency due to reduced compatibility with the ribosome's catalytic active site and nascent peptide exit tunnel. As described above, other facets of the translation system (tRNAs, EF-Tu, etc.) must be tuned to facilitate the use of such monomers. Below, we discuss recent advances that set the stage to engineer the ribosome itself, which include methods for purification, reconstitution, synthesis, engineering, and evolution of the ribosome in vitro. ## Ribosome reconstitution The ribosome is composed of a small (30S) and large (50S) subunit. The 30S subunit decodes mRNA and accommodates corresponding tRNA-monomers. The 50S subunit accommodates tRNA-monomers, catalyzes polypeptide synthesis, and excretes polypeptides. Structure, function and assembly studies of the ribosome have transformed our understanding of the ribosome's parts, defined functional relevance, and elucidated mechanism. This has been greatly facilitated by ribosome reconstitution and purification approaches. Traditional methods for isolating ribosomes for testing in vitro, including mutant versions, involve purification via ultracentrifugation and supplementation of in vivosynthesized ribosomes or ribosomal RNA (rRNA) into expression reactions. While this methodology allows a great deal of study into ribosome structure and function, it has been limited in its ability to generate some mutant rRNAs, which result in lethality in the cells which depend on them for protein synthesis. To circumvent this limitation, several groups have developed strategies for plasmid expression and purification of affinitytagged rRNA variants to circumvent this difficulty, enabling in vitro characterization of otherwise inaccessible mutants. Similar methods were later used to isolate ribosomes evolved for orthogonal templates and enhanced genetic code expansion for testing in vitro. These ribosomes may be directly supplemented into in vitro translation reactions for dissection of ribosome function, or the rRNA Nucleic Acids and protein components can be isolated and recombined in ribosome reconstitution experiments to study ribosomal subunit assembly. ## 30s subunit reconstitution. As an alternative to building ribosomes in cells, followed by their purification, ribosomes can be assembled, or reconstituted from in vivo purified components or in vitro synthesized parts. In a groundbreaking paper, Taub and Nomura demonstrated for the first time that isolated 16S rRNA and the total complement of 30S ribosomal proteins (TP30) were sufficient for reconstitution of functional E. coli 30S particles in a single step. This simple strategy, when coupled with advancing imaging techniques, was enough for the elucidation of much of the 30S assembly process. Initial assessments of relationships between ribosomal proteins in the structure of the ribosome were determined via chemical iodination studies. It was later shown that 30S particles could be assembled from TP30 and in vitro synthesized 16S rRNA lacking base modifications, demonstrating that these modifications are not required for 30S assembly. These particles formed 70S ribosomes when supplied with purified 50S particles which were capable of A-and P-site binding, but not peptidyl transferase activity. Ribosome biogenesis factors play an important role in the assembly of functional ribosomes in vivo and have been shown to facilitate 30S assembly in vitro. Leveraging this knowledge, Tamaru et al. were able to synthesize 30S particles from 16S rRNA and 30S r-proteins all individually purified from cells and assembled in the presence of ribosome biogenesis factors and under physiological conditions. Assembly of 30S particles from independently generated components can provide exquisite control over assembly and function of the translation machinery, facilitating engineering applications. Li et al. achieved this milestone by synthesizing 30S particles from fully in vitro generated 16S rRNA and TP30 supplemented 30S ribosome assembly factors to assemble 30S particles with 21% activity of native ribosomes. Taken together, recent advances in 30S reconstitution have begun the march towards building ribosomes de novo in test tubes, as well as mutant ribosomes capable of enhanced synthesis of proteins with ncAAs. 50S subunit reconstitution. While significant strides have been made in bottom-up synthesis of 30S subunits, in vitro generation of active 50S particles has posed a greater challenge. Reconstitution of the 50S ribosomal subunit was first achieved by Nierhaus and Dohme, but required a twostep incubation at high salt concentration and temperature. Despite the non-physiological nature of this reconstitution, this methodology combined with scanning transmission electron microscopy was instrumental in elucidating both the order and dependencies of r-protein binding and 50S assembly. However, 50S subunits reconstituted in this way are less active than those purified from cells. Single-step reconstitution of large subunits from the halophilic Haloferax mediterranei has been achieved in the presence of high (2.5 M) monovalent cation and 60 mM magnesium concentrations at the optimal growth temperature (40-45 - C) for this organism, indicating that this two-step requirement is not universal across diverse organisms. In generating mutant 50S particles, it is desirable to use in vitro transcribed 23S rRNA, whereby mutations can be easily introduced without cell-viability concerns. In vitro transcription and methylation studies of the E. coli 23S rRNA from a linearized template using T7 RNA polymerase were the first step toward synthetically produced large ribosomal subunits. Unfortunately, rRNA produced in this way does not assemble correctly into E. coli 50S particles -a deficit which was attributed to a lack of critical base modifications to the synthetic transcript which are present in wildtype 23S. However, in vitro transcribed rRNA from two thermophiles were able to be assembled into functional large subunits in a one-or two-step treatment with highsalt, indicating that these modifications are not in fact essential for ribosome function. Later experiments demonstrated that the addition of osmolytes to in vitro transcribed reconstitution reactions of E. coli 23S rRNA resulted in a 100-fold improvement in the assembly of active large subunits (179). However, 50S particle reconstitution still relies on 50S proteins purified from cells -assembly of 50S particles from fully in vitro synthesized parts as was achieved with the 30S in (171) has not yet been reported. ## Integrated ribosome synthesis and assembly The ability to reconstitute both the small and the large subunit of the ribosome from in vitro transcribed RNA suggested that a fully integrated approach to ribosome assembly might be possible. In developing such a method, it was important to identify conditions which would promote the assembly of both subunits under similar, ideally physiological, conditions. Carrying out transcription of rRNA in a cell extract which mimicked the cytoplasm (180) was identified as a promising strategy to enable assembly of both subunits under similar, physiological conditions. Indeed, by expressing the entire rrnB operon under the control of the T7 promoter in ribosome-depleted cell extract supplemented with TP70, integrated synthesis, assembly, and translation (iSAT) of a reporter protein was demonstrated in a single reaction. This method enables synthesis and testing of ribosomes that would be dominant lethal or otherwise inviable in vivo. Further optimization of the genetic construct encoding the ribosomal operon yielded a 45-fold improvement in ribosome activity in iSATand crowding and reducing agents conferred a further ∼4-fold improvement. Carrying out iSAT in a semicontinuous reaction vessel to allow diffusion of waste products and energy sources yielded another 7-fold improvement in protein yields. An evolutionary design of experiment approach was able to identify further optimizations to reduce reagent use as well as implement a less costly energy source -an approach which holds promise for further optimizations of the platform (31). ## Ribosome evolution and engineering While capabilities are emerging to build ribosomes from the ground up, parallel efforts have reached important milestones for engineering extant ribosomes. Key among these is atomic mutagenesis, an elegant technique pioneered by the Polacek lab which uses in vitro assembly of 50S particles to introduce atomic mutations and other non-canonical modifications into 23S rRNA (reviewed in (184)). Leveraging classical ribosome complementation assaysand employing chemically synthesized RNA oligonucleotides as the complementing fragment, researchers may incorporate bases with atomic level modifications to dissect the role of individual nucleobases in various aspects of ribosome function. This method has been used to elucidate the role of specific nucleotides in peptide bond formation, EF-G triggered GTP hydrolysis, and tRNA binding. Atomic mutagenesis of mRNA has also been utilized to elucidate the mechanism of stop codon recognition by release factors. Looking forward, this approach appears to be promising for constructing a new generation of engineered ribosomes whose chemical activity and substrate specificity can be augmented by adding artificial nucleotides into 23S rRNA. Towards evolution of the ribosome for new functions, several projects to date have pursued ribosome directed evolution using in vivo systems, and have enjoyed noteworthy successes such as ribosomes which are orthogonaland release factor resistant, or capable of quadruplet decoding, or ␤-amino acid incorporationamong others. Unfortunately, such efforts are constrained by the fact that cell-viability limits the changes that can be made to the ribosome, with many mutations conferring dominant lethal phenotypes. Recently, Orelle et al. addressed this gap through the construction of the first complete functionally orthogonal ribosome-mRNA system in cells, where a sub-population of ribosomes are available for engineering and are independent from wild-type ribosomes supporting cell life. This was achieved by constructing a ribosome with covalently tethered subunits called Ribo-T (the core 16S and 23S ribosomal RNAs form a single chimeric molecule with the connection at where helix h44 of the 16S rRNA and helix H101 of the 23S rRNA). Ribo-T was evolved by selecting otherwise dominantly lethal rRNA mutations in the PTC that facilitate the translation of problematic protein sequences not accessible to natural ribosomes. Similar tethered, or stapled ribosomes, were used to access new ribosome function, as well as incorporate ncAAs. Unfortunately, covalently linked ribosomes appear to have reduced rates of ribosome assembly, initiation, and termination. Despite this, they serve as a key first step to being able to engineer the large subunit of the ribosome towards the polymerization of monomers with non-canonical backbones. Curiously, despite the development of an in vitro ribosome selection strategy 15 years ago, little ribosome evolution work has been done in vitro since that time. Perhaps this is because the ribosomes selected in this study were still produced from an in vivo produced library, and therefore performing the selection in vitro represents an unnecessary added complexity. However, there are advantages to performing selections in vitro, including a greater degree of control over selection conditions, the potential for larger library sizes, and enhanced throughput. The development of the ability to synthesize functional ribosomes in vitro in the iSAT system may make in vitro selection of the ribosome more attractive, and forthcoming work will set the stage for the development of this technology. To advance this mission, in vitro encapsulation may prove useful. ## Conclusions and future directions Looking forward, we anticipate that continued developments of in vitro translation system engineering will have fundamental importance and significant practical applications. First, new research will help shed light on the intricacies of bacterial translation. Translation is a highly complex system, and just as successful examples of engineering the translation apparatus illustrate our understanding of it, instructive failures reveal gaps in our current understanding. By engineering bacterial translation to accommodate novel monomers, we will dramatically perturb each step of translation and allow new biochemical analysis of individual steps. This fundamental knowledge has been previously difficult to obtain with available wild-type translation components and traditional mutagenesis strategies, and is still constrained in cells by viability concerns. Beyond fundamental scientific breakthroughs in the biology of translation, new advances could also inform our understanding of life's origins. According to the RNA World hypothesis, protein translation, especially the origin and evolution of the ribosome, shepherded the transition from the primordial biota relying on RNA to the modern world. The high conservation of the catalytic core of the ribosome raises fundamental questions about the origin and evolution of the translation system. Understanding the structural and substrate flexibility of the ribosome may provide important experimental insights that are currently missing. With respect to emerging opportunities, the ability to produce peptides and polymers comprised of only nonproteinogenic monomers could lead to new applications based on novel sequence-defined polymers, such as new classes of peptidomimetic drugs or advanced materials. Just as organic chemistry once revolutionized the ability of chemists to build molecules following a basic set of design rules, so too will new foundational technologies for the biosynthesis of sequence defined polymers from engineered translation machinery provide a transformative toolbox for synthetic and chemical biology.